JP2022512740A - Recombinant ingredients and compositions for use in food products - Google Patents

Abstract

Methods for producing food products containing recombinant components, as well as compositions used in such methods and food products produced by such methods are provided.

Description

The present invention generally relates to alternatives to animal-derived food products. In particular, the present invention relates to recombinant components and compositions for use in food products, methods for producing these components and compositions, and methods for producing food products containing these components and compositions. Regarding.

Related Applications This application claims priority to US Patent Application No. 62 / 746,918, filed October 17, 2018, which is incorporated herein by reference in its entirety. Is done.

Animal-derived food products (eg meat, milk, eggs) are a popular source of nutrition. Animal-derived food products include high-quality proteins, essential minerals (eg, calcium, phosphorus, zinc, magnesium), and vitamins (eg, riboflavin, vitamin A, vitamin B12). In addition, many such food products have advantageous functional properties that allow the production of a wide variety of derivative food products (eg yogurt, cheese, cream, ice cream, butter, mayonnaise).

However, animal-derived food products contain components that can cause unhealthy reactions in humans (eg, lactose, allergens, saturated fats, cholesterol). In addition, the production of these food products has significant impacts on animal welfare and the environment, including livestock with the potential for contamination by pathogens, pesticide residues, heavy metals, and aflatoxin M1.

These concerns have encouraged the development of alternatives to animal-derived food products. Some such alternatives include plant-based components (eg, proteins, lipids, vitamins). However, alternatives to animal-derived food products are increasingly produced from components that are recombinantly produced (eg, using microbial host cells) (eg, proteins, lipids).

The use of recombinant ingredients in food products poses new problems. One such problem is that food products produced from recombinant components contain significant amounts of such recombinant components (more than was typical in previous products in which recombinant components were utilized). Often, the use of large amounts of recombinant components is the fact that they can be affected by other potentially unwanted components that are simultaneously produced by the microbial host cell that produces the recombinant component.

One such other component is esterase activity. Esterases can hydrolyze ester bonds in diglycerides, triglycerides, phospholipids, and / or lipoproteins to release free fatty acids. Compositions comprising diglycerides, triglycerides, phospholipids, and / or lipoproteins are used in various food products for which recombinant components may be used. In some such food products, esterase-catalyzed release of free fatty acids can have detrimental effects on properties such as texture, emulsification, aroma, flavor, and nutrient content. In other such food products, esterase-catalyzed release of free fatty acids can have beneficial effects on properties such as, for example, flavor profiles (eg, flavor profiles of aged cheese).

As a result, challenges must be overcome in the production, processing, and use of recombinant components for the production of animal-derived food product substitutes, especially with respect to esterase activity.

Exemplary embodiments will be described and described by use of the accompanying drawings, along with additional specificity and details.

PCT filing PCT / US2015 / 046428 PCT filing PCT / US2017 / 48730 International Publication No. 96/29391 Pamphlet

Howksworth et al. , In, Ainthworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK Pearson. 1990. Methods Enzymol. 183: 63 Altschul et al. 1990. J. Mol. Biol. 215: 403 Maryshev et al. 2014. Nature 509: 385 Li et al. 2014. J. Am. Chem. Soc. 136: 826 Deshpanda et al. 2008. Glycobiology 18 (8): 626 Kavir & Kazi. 2011. Genet Mol Biol. 34 (4): 587

Therefore, there is a need for methods that can be used to produce alternatives to animal-derived food products from recombinant components, as well as compositions that are used and obtained from such methods.

In one aspect, it is a method for producing a food product containing a recombinant component produced by a recombinant microbial host cell capable of producing the recombinant component, wherein the activity of the esterase is essentially eliminated or A method comprising at least one step to be modulated is provided herein.

In another aspect, a preparation comprising a recombinant component and an esterase activity that is essentially eliminated or modulated relative to the esterase activity contained in the corresponding preparation is provided herein.

In another embodiment, a recombinant microbial host cell capable of producing a recombinant component and comprising an esterase activity essentially eliminated or modulated as compared to the esterase activity contained in the corresponding recombinant microbial host cell. Is provided herein.

In another aspect, a food product containing a recombinant component produced by a recombinant microbial host cell, which is essentially eliminated or modulated relative to the activity of esterase in the corresponding food product. Food products further comprising the activity of esterase are provided herein.

In the chart showing the results of the free fatty acid (FFA) assay of recombinant β-lactoglobulin preparations obtained from purification and removal of esterase activity based on charge (ie, ionic complex formation) and thermal instability / thermal stability. The "final material" is the preparation after removing the high concentration of salt and exchanging the buffer to concentrate the recombinant β-lactoglobulin according to various typical embodiments of the present invention. .. FIG. 6 shows a stained native PAGE gel of a recombinant β-lactoglobulin preparation obtained by purifying and removing esterase activity based on charge (ie, ionic complex formation) and thermal instability / thermal stability. The "final material" is the preparation after removal of high concentrations of salt and replacement of buffer to concentrate recombinant β-lactoglobulin according to various representative embodiments of the invention. It is a chart showing the result of the FFA assay of the recombinant β-lactoglobulin preparation obtained by purifying and removing the esterase activity based on charge (ie, ionic complex formation) and hydrophobicity, and the "final material" is: The preparation after removal of high concentrations of salt and replacement of buffer to concentrate recombinant β-lactoglobulin according to various representative embodiments of the invention. FIG. 6 shows a stained native PAGE gel of a recombinant β-lactoglobulin preparation obtained by purifying and removing esterase activity based on charge (ie, ionic complex formation) and hydrophobicity, “final material”. However, according to various representative embodiments of the present invention, the preparation is made after removing the high concentration of salt and exchanging the buffer solution to concentrate the recombinant β-lactoglobulin. It is a diagram showing the result of the pNPL assay using the Chuky Kramer analysis of purified fermented broth obtained from the culture containing the indicated defoaming agent according to various representative embodiments of the present invention. FIG. 6 is a map of the target vector used to produce recombinant host cells containing the removed cutinase activity according to a representative embodiment of the invention. A set obtained from a recombinant host cell containing the depleted cutinase activity compared to a recombinant β-lactoglobulin preparation obtained from the corresponding recombinant host cell according to various representative embodiments of the invention. 6 is a chart showing the results of the FFA assay for recombinant β-lactoglobulin preparations. A set obtained from a recombinant host cell containing the depleted cutinase activity compared to a recombinant β-lactoglobulin preparation obtained from the corresponding recombinant host cell according to various representative embodiments of the invention. FIG. 5 shows a stained native PAGE gel of a recombinant β-lactoglobulin preparation. Photographs of gel compositions of frozen confectionery containing recombinant or natural bovine β-lactoglobulin in the presence or absence of esterase activity according to various representative embodiments of the present invention are shown. Photographs of gel compositions of frozen confectionery containing recombinant or natural bovine β-lactoglobulin in the presence or absence of esterase activity according to various representative embodiments of the present invention are shown. Photographs of gel compositions of frozen confectionery containing recombinant or natural bovine β-lactoglobulin in the presence or absence of esterase activity according to various representative embodiments of the present invention are shown. Photographs of gel compositions of frozen confectionery containing recombinant or natural bovine β-lactoglobulin in the presence or absence of esterase activity according to various representative embodiments of the present invention are shown. Photographs of gel compositions of frozen confectionery containing recombinant or natural bovine β-lactoglobulin in the presence or absence of esterase activity according to various representative embodiments of the present invention are shown. Photographs of gel compositions of frozen confectionery containing recombinant or natural bovine β-lactoglobulin in the presence or absence of esterase activity according to various representative embodiments of the present invention are shown.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Further, unless the context specifically requires, the singular term shall include the plural and the plural term shall include the singular.

Definitions The terms "one (a)" and "one (an)" and "the" and similar references used herein are not otherwise indicated or contextual. Both singular and plural, if not clearly inconsistent.

As used herein, the term "about" refers to being within an acceptable margin of error for a particular value determined by one of ordinary skill in the art, which is how the value is. It may be measured or determined, or it may depend to some extent on the limits of the measurement system.

As used herein, the term "and / or" refers to a plurality of components that are combined or excluded from each other. For example, "x, y, and / or z" is "x" alone, "y" alone, "z" alone, "x, y, and z", "(x and y) or z", or " It can refer to "x or y or z".

As used herein, the term "corresponding preparation" refers to an esterase in which the esterase activity contained in the "corresponding preparation" (eg, the preparation is essentially removed or modulated). Contains recombinant microbial host cells that contain activity and / or recombinant microbial host cells that have been cultured under conditions suitable to essentially eliminate or modulate esterase activity, and / or esterase inhibitors. Not essentially removed or modulated as provided herein (because it is not derived from fermented broth or because the recombinant component has not been purified to remove esterase activity). Refers to a preparation that is identical to the preparation compared to the "corresponding preparation", except for.

The term "corresponding food product" as used herein is essentially esterase activity as the method used to produce the "corresponding food product" is provided in the method provided herein. Produced by the same method used to produce a food product compared to the "corresponding food product", except that it does not include at least one step that is removed or modulated into. Refers to food products.

The term "corresponding recombinant microbial host cell" is essentially eliminated so that the esterase activity of the "corresponding recombinant microbial host cell" is provided in the recombinant microbial host cell provided herein. Refers to a recombinant microbial host cell that is identical to a recombinant microbial host cell compared to a "corresponding recombinant microbial host cell", except that it is neither modulated nor modulated.

As used herein, the terms "essentially absent" and "essentially removed" mean that the indicated component is not detectable in the indicated composition by common analytical methods. Or the indicated component is present in trace amounts that are not functional. As used in this context, the term "functional" means that it does not contribute to the properties of a composition that contains trace amounts of the indicated ingredients, or that it is active in the indicated composition that contains trace amounts of the indicated ingredients (eg,). , Enzyme activity), or has no adverse health effects on the consumption of compositions containing trace amounts of the indicated ingredients.

As used herein, the term "esterase activity" or "esterase activity" refers to the activity of an enzyme capable of hydrolyzing an ester bond (ie, a hydrolase that acts on the ester bond). Esterase has an enzyme number (EC number) 3.1. Indicated by. The terms are used interchangeably herein.

As used herein, the term "fermented broth" refers to a culture containing recombinant microbial host cells capable of producing recombinant components.

As used herein, the term "filamentous fungal cell" is a cell derived from any filamentous morphology of subdivided fungi and the phylum Oomycete (defined by Non-Patent Document 1). Is. Filamentous mycelial cells are distinguished from yeast by the elongation of their hyphae during vegetative growth.

As used herein, the term "fungus" refers to ascomycetes, basidiomycetes, zygomycota, and chytrids, phylums, and glomeromycota. However, it is understood that fungal taxonomy is constantly evolving and therefore this particular definition of the fungal kingdom may be adjusted in the future.

As used herein, the term "heterogeneous" means that it does not normally exist in the context in which it is described. In other words, an entity thus characterized is exogenous in the context in which it is described. When used in connection with a protein produced by filamentous fungal cells, the term implies that the protein is not naturally produced by filamentous fungal cells.

As used herein, the term "host cell" refers to a cell into which recombinant nucleic acid has been introduced. It should be understood that such terms are intended to refer not only to specific target cells, but also to the progeny of such cells. Such progeny may not actually be identical to the parent cell, as they may occur in generations that are followed by certain alterations due to mutations or environmental influences, but are still used herein. Included within the term "host cell".

As used herein in the context of a polynucleotide or polypeptide sequence, the term "identical" refers to a residue in the reference window of two sequences that are identical when aligned for maximum matching. There are several different algorithms known in the art that can be used to measure the identity of nucleotide or protein sequences. For example, the sequences are FASTA (eg, provided in Wisconsin Genetics Software Package Version 10.0, Genetics Computer Group, Madison, WI), GAP (eg, Wisconsin Genetics Software , Provided to WI), BESTFIT (eg, Provided to Wisconsin Genetics Software Package Version 10.0, Genetics Computer Group, Madison, WI), CrustalW (eg, provided to WI), using the default parameters of version 1.83. Or can be compared using BLAST (eg, using mutual BLAST, PSI-BLAST, BLASTP, BLASTN; eg, provided through the National Center for Biotechnology Information) (eg, non-patentable). Document 2; see Non-Patent Document 3). For alignment by these methods, default parameters may be used (eg, in BLASTN, the default parameters are Gap opening penalty = 5 and Gap extension penalty = 2, and in BLASTP, the default parameters are Gap opening penalty = 11 and Gap. extension penalty = 1). The reference window can range from the full length of the two sequences to only the respective region of the two sequences (eg, a region that is at least 50 nucleotides or at least 10 amino acids long).

As used herein, the terms "include", "includes", "having", "has", "with", or variants thereof are referred to as the term "inclusive". It is intended to be inclusive to resemble "comprising".

As used herein, the term "microbe" is an abbreviation for microorganism and refers to a unicellular organism. As used herein, the term includes all bacteria, paleontology, unicellular protists, unicellular animals, unicellular plants, unicellular fungi, unicellular algae, protozoa, and chromista organisms.

As used herein in relation to esterase activity, the term "modulated" is one of the activity of esterase, including, for example, any one disclosed herein, including an increase or decrease in the activity of the esterase. Any modification of the activity of an esterase or the activity of any combination of at least two esterases disclosed herein. Such modulated esterase activity is typically due to an increase or decrease in esterase concentration or an increase or decrease in esterase enzyme activity.

As used herein, the term "natural" refers to what is found in nature.

As used herein, the term "as needed" or "as needed" means the presence or absence of features or structures, or the occurrence or absence of events or situations. The description includes the case where a specific feature or structure exists and the case where the feature or structure does not exist, or the case where an event or situation occurs, and the case where the event or situation does not occur.

As used herein, the term "polynucleotide" refers to RNA, cDNA, both sense and antisense strands of genomic DNA, as well as the synthetic and mixed polymers described above. Polynucleotides may be chemically or biochemically modified, or may contain unnatural or inducible nucleotide bases. Such modifications include, for example, labeling, methylation, substitution with one or more analogs of naturally occurring nucleotides, uncharged linkages (eg, methylphosphonates, phosphotriesters, phosphoramidates). , Carbamate), charged bonds (eg, phosphorothioates, phosphorodithioates), pendant moieties (eg, polypeptides), intercalators (eg, acridine, solarene), chelators, alkylating agents, and modified bonds (eg, eg Includes alpha anomaly nucleic acid). Examples of modified nucleotides are known in the art (see, eg, Non-Patent Document 4; Non-Patent Document 5). Also included are synthetic molecules that mimic polynucleotides with their ability to bind to specified sequences via hydrogen bonds and other chemical interactions. Such molecules are known in the art and include, for example, molecules in which peptide bonds substitute for phosphate bonds in the skeleton of the molecule. Other modifications can include analogs containing other structures, such as modifications in which the ribose ring is found in cross-linked moieties or "locked" polynucleotides.

As used herein, the term "preparation" refers to a preparation obtained when the recombinant component is separated from one or more other components of the fermented broth.

As used herein, the term "protein" refers to the polymer form of an amino acid of any length, which is a coding and non-coding amino acid, a naturally occurring amino acid and a non-naturally occurring amino acid. Amino acids that have been chemically or biochemically modified or derivatized, as well as polypeptides with a modified peptide backbone can be included.

As used herein, the term "purify" means that a recombinant protein is substantially separated from the cellular components of a recombinant microbial host cell (eg, membrane lipids, chromosomes, proteins). The term does not require (if any) that the recombinant protein is separated from all other chemicals.

As used herein, the term "recombinant microbial host cell" refers to a microbial cell containing a recombinant polynucleotide. It should be understood that such terms are intended to refer not only to specific target cells, but also to the progeny of such cells. Such progeny may not be identical to the parent cell, as certain modifications may occur in subsequent generations due to mutations or environmental influences, but the term still used herein. Included within the scope of "recombinant microbial host cells".

As used herein, the term "recombinant polynucleotide" refers to a polynucleotide that has been removed from its naturally occurring environment, a polynucleotide that borders or is in close proximity to a polynucleotide as it is found naturally. A polynucleotide that is not associated with all or part of it, a polynucleotide that is operably linked to a polynucleotide that is not linked in nature, or a polynucleotide that does not exist in nature. The term can be used, for example, to describe a polynucleotide containing a cloned DNA isolate or a chemically synthesized nucleotide analog. A polynucleotide is also considered a "recombination" if it contains any non-naturally occurring genetic modification. For example, an endogenous polynucleotide is a "recombinant polynucleotide" if it contains, for example, an insertion, deletion, or substitution of one or more nucleotides artificially introduced by human intervention. Is considered to be. Such modifications include point mutations, substitution mutations, deletion mutations, insertion mutations, missense mutations, frameshift mutations, duplicate mutations, amplification mutations, translocation mutations, or inversions to polynucleotides. Mutations can be introduced. The term includes polynucleotides in the chromosomes of host cells, as well as polynucleotides that are not on the chromosomes of host cells (eg, polynucleotides contained in episomes).

As used herein, the term "recombinant protein" is produced in cells of a different species or type when compared to the species or type of cell that produces the protein in nature, or it is not produced in nature. A protein produced in cells at a level.

As used herein, the term "vector" is a nucleic acid molecule capable of transporting another nucleic acid to which a nucleic acid is linked. One type of vector is a "plasmid", which generally refers to a circular double-stranded DNA loop that may be ligated with additional DNA segments, either from or restricted by polymerase chain reaction (PCR) amplification. It also includes linear double-stranded molecules such as molecules resulting from the treatment of cyclic plasmids with enzymes. Other vectors include cosmids, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs). Another type of vector is a viral vector, in which additional DNA segments may be ligated into the viral genome (discussed in more detail below). Certain vectors can proliferate autonomously in the host cell into which they are introduced (eg, a vector with an origin of replication that functions in the host cell). Other vectors can be integrated into the host cell's genome when introduced into the host cell, thereby replicating with the host genome.

As used herein, the term "yeast" refers to organisms of the order Saccharomyces, such as Saccharomyces cerevisiae and Pichia pastoris. Nutrient growth of yeast is caused by budding / vesicle formation of unicellular fronds, and carbon catabolism may be fermentable.

Methods for Producing Food Products Containing Recombinant Components In one embodiment, recombinant microbial host cells capable of producing recombinant components (eg, any of the recombinant microbial host cells provided herein). Provided is a method for producing a food product containing a recombinant component produced by (eg, any of the recombinant components disclosed herein), wherein the method provides esterase activity (ie, esterase activity). At least one in which (eg, the activity of any one esterase disclosed herein or the activity of any combination of at least two esterases disclosed herein) is essentially eliminated or modulated. Includes one step.

In some embodiments, the invention is disclosed herein based on the finding that recombinant microbial host cells producing recombinant components also typically also produce esterase activity (eg, herein). Provided is one or more methods for producing a food product containing any of the recombinant components). Such esterase activity can be co-purify with the recombinant component. When the recombinant component is then used to produce a food product, the esterase activity hydrolyzes the ester bonds in the diglycerides, triglycerides, phospholipids, and / or lipoproteins contained in the food product, thus It can catalyze the production of free fatty acids in food products.

As mentioned above, the production of free fatty acids in food products produces, for example, unpleasant aromas and / or tastes, prevents the formation of emulsions, has an undesired effect on texture, and is an essential nutrient (eg, vitamins). May have harmful effects by interacting with, thereby reducing nutrient content and reducing shelf life. The production of free fatty acids in food products can also have beneficial effects, for example, by producing the desired flavor profile (eg, flavor profile of aged cheese) or by making enzyme-treated cheese for use in processed cheese. be.

In some embodiments, the invention is based on the finding that the esterase activity produced by recombinant host cells is different from the esterase activity contained in milk produced from mammals (eg, the present specification). Provided is one or more methods for producing a food product containing any of the recombinant components disclosed in this document.

Accordingly, various embodiments of the invention include specific methods for essentially eliminating or modulating the esterase activity produced by recombinant microbial host cells. By essentially eliminating or modulating the esterase activity in food products containing recombinant components produced by recombinant microbial host cells, the present invention inhibits or delays the production of unpleasant odors and tastes, milk. It provides methods for forming turbidity, increasing nutrient content, modulating texture, extending shelf life, and / or producing the desired flavor profile in food products more quickly.

In some embodiments, the methods provided herein for producing a food product include at least one step in which esterase activity is essentially eliminated.

In some embodiments, the methods provided herein for producing a food product include at least one step of reducing esterase activity. In some such embodiments, the esterase activity is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%. At least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 %, Or 100% decrease.

In some embodiments, the methods provided herein for producing a food product include at least one step of increasing esterase activity. In some embodiments, esterase activity is at least 25%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%. , At least 700%, at least 800%, at least 900%, or at least 1,000%.

In some embodiments, the methods provided herein for producing a food product include at least one step of essentially eliminating the first esterase activity and reducing the second esterase activity. .. In some embodiments, the methods provided herein for producing a food product include at least one step of essentially eliminating the first esterase activity and increasing the second esterase activity. .. In some embodiments, the methods provided herein for producing a food product include at least one step of increasing the first esterase activity and decreasing the second esterase activity.

In some embodiments, the methods provided herein for producing a food product are the activity of an esterase (eg, the activity of any one esterase disclosed herein or disclosed herein. The activity of any combination of at least two esterases) is essentially eliminated or comprises one step.

In some embodiments, the methods provided herein for producing a food product are the activity of an esterase (eg, the activity of any one esterase disclosed herein or disclosed herein. The activity of any combination of at least two esterases is essentially eliminated or modulated by two or more steps (eg, 2 steps, 3 steps, 4 steps, 5 steps) (eg, 2 steps, 3 steps, 4 steps, 5 steps). , Essentially eliminated or modulated esterase activity, eg, the activity of any one esterase disclosed herein or any combination of at least two esterases disclosed herein. Includes two or more steps selected from the steps disclosed herein provided.

Esterase activity in the manner provided herein for producing food products that essentially eliminate or modulate esterase activity (eg, the activity of any one esterase disclosed herein or herein. At least one step in which (the activity of any combination of at least two esterases disclosed in the book) is essentially eliminated or modulated provides essentially eliminated or modulated esterase activity. Any step can be included.

In some embodiments, esterase activity (eg, activity of any one esterase disclosed herein or disclosed herein) in the manner provided herein for producing food products. At least one step in which the activity of any combination of at least two esterases) is essentially eliminated or modulated is recombinant under conditions appropriate to essentially eliminate or modulate the esterase activity. It comprises culturing recombinant microbial host cells capable of producing the component.

Thus, in some such embodiments, the methods provided herein can a) produce a recombinant component (eg, any of the recombinant components disclosed herein). Steps to obtain recombinant microbial host cells; b) culture medium under conditions suitable for essentially removing or modulating esterase activity, as well as recombinant components and essentially removed or modulated esterase. Steps of culturing recombinant microbial host cells in a culture medium under conditions suitable for the production and / or secretion of recombinant components to obtain active fermentation broth; c) Recombinant components from fermentation broth as needed. Purification to obtain a preparation containing recombinant components and essentially removed or modulated esterase activity; and d) recombinant components and essentially removed or modulated esterase activity. Includes steps to prepare a food product from a fermented broth or preparation containing.

Suitable conditions for culturing recombinant microbial host cells in a culture medium under conditions suitable for essentially removing or modulating esterase activity are, for example, under which the recombinant microbial host cells have esterase activity. A condition that essentially eliminates or modulates its generation of. Non-limiting examples of such conditions are the right pH, the right temperature, the right feed, the right pressure, the right nutrient content (eg, the right carbon content, the right nitrogen content, the right phosphorus). Content), the appropriate dietary supplement content, the appropriate trace metal content, and / or the appropriate oxygenation level. In some embodiments, the appropriate pH at which the recombinant microbial host cell essentially eliminates or modulates its production of esterase activity is 2 and 7.5, 6.5, 6, 5.5, 5. , 4.5, 4, 3.5, 3, or 2.5; 2.5 and 7.5, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, Or between 3; between 3 and 7.5, 6.5, 6, 5.5, 5, 4.5, 4, or 3.5; between 3.5 and 7.5, 6.5, 6, Between 5.5, 5, 4.5, 4; between 4 and 7.5, 6.5, 6, 5.5, 5, 4.5; between 4.5 and 7.5, 6.5, Between 6, 5.5, 5; between 5 and 7.5, 6.5, 6, 5.5; between 5.5 and 7.5, 6.5, 6; between 6 and 7.5, Between 6.5; between 6.5 and 7.5, or between 7, or between 7 and 7.5. In some embodiments, the appropriate dietary supplement content in the culture medium in which the recombinant microbial host cell essentially eliminates or modulates its production of esterase activity is an antifoaming agent. Non-limiting examples of suitable antifoaming agents are Struktol J 673 A (Schill & Seilacher GmbH, Hamburg, Germany), Industrol DF204 (BASF Canda, Inc., Missisusauga, Canda, Poly), Poly. ), Hodag K-60K (Hodag Momentive Corp., Chicago, IL), and Eroll DF6000K (PMC Own, Carvin, France), ACP 1500 (Dow Momentive Company, MidLandSi) , MO), SAG 471 (Momentive Performance Materials Inc., Waterford, NY), SAG 5693 (Momentive Performance Material Materials Inc., WaterFord, NY), SAG 710 (Moment) Includes Performance Momentives Inc., Waterford, NY), silicone defoamers, Struktor J647 (Schill & Selicher, Hamburg, Germany), and sunflower oil.

In some embodiments, esterase activity (eg, activity of any one esterase disclosed herein or disclosed herein) in the manner provided herein for producing food products. The activity of any combination of at least two esterases) is essentially eliminated or modulated by at least one step, fermentation obtained from a culture of recombinant microbial host cells capable of producing recombinant components. Includes steps to essentially eliminate or modulate esterase activity in the broth or preparation.

Thus, in some such embodiments, the methods provided herein can a) produce a recombinant component (eg, any of the recombinant components disclosed herein). Steps to obtain recombinant microbial host cells; b) Culturing the recombinant microbial host cells in a culture medium under conditions suitable for the production and / or secretion of the recombinant components to obtain a fermentation broth containing the recombinant components; c) Steps to purify the recombinant component from the fermented broth as needed to obtain a preparation containing the recombinant component; d) Recombined by essentially removing or modulating the esterase activity in the fermented broth or preparation. Steps to obtain a fermented broth or preparation containing components and essentially removed or modulated esterase activity; and e) fermented broth containing recombinant components and essentially removed or modulated esterase activity. Or it involves the step of preparing a food product from the preparation.

Esterase activity in the fermentation broth or preparation can be essentially eliminated or modulated, for example, by adding an esterase inhibitor to the fermentation broth or preparation. Non-limiting examples of suitable esterase inhibitors include synthetic inhibitors (eg, phosphonates, boronic acid, lipid analogs) and, for example, Juniperus communis, Illicium religiosum, Panax japonicus rhizomes. ), Panax ginseng, American carrot (Panax quinquefolius), Siberian ginseng (Acanthopanax senticosus), Chanoki variant Sinensis (Camellia sinensis var. bellflower (Platycodi radix), Salacia reticulata (Salacia reticulate), lotus (Nelumbo nucifera), down Liu willow (Salix matsudana), Narukobie (Eriochloa villosa), Salacia reticulata, high Matsumushisou (Scabiosa tschiliensis Grun), and the Man Shu Acanthopanax (Acanthopanax sessiliflorus) Natural inhibitors isolated from sources such as (eg, β-lactone (eg, varilactone, evelactone A & B, and vibralactone), manno-oligosaccharides, acetylcholineresterase inhibitors, cholinesterase inhibitors, polyphenols, saponins, panax japonicus, etc. Includes hesperidin, kaurelpenin, proanthocyanidin, orristat (Xenical), carnosic acid, estin, crosin, crocetin, chlorogenic acid, neochlorogenic acid, ferroylquinic acid, e-polylysine, chitosan, chitinic acid). In some such embodiments, the esterase inhibitor requires the addition of a pH and / or ionic strength modifier for activity.

The esterase activity in the fermentation broth or preparation is, for example, removing from the fermentation broth or preparation the cofactors required for the activity of the esterase and / or the cofactors required for the activity of the esterase inhibitor and / or the fermentation broth or preparation. It can also be essentially removed or modulated by addition to. Non-limiting examples of such cofactors include metals (eg, divalent cations such as calcium). In some embodiments, the divalent cation (eg, calcium) is removed by binding the divalent cation to the chelating agent. A non-limiting example of a suitable chelating agent is ethylenediaminetetraacetic acid (EDTA).

The esterase activity in the fermentation broth or preparation can also be essentially removed or modulated, for example by heat or non-heat treatment. Non-limiting examples of heat treatment include pasteurization and sterilization. Non-limiting examples of non-heat treatment include high pressure pasteurization (ie, high pressure treatment, HPP), sonication, pulsed electric field treatment, and irradiation. In some embodiments, the esterase activity in the fermented broth or preparation is by incubating the fermented broth or preparation at high temperature for a relatively short period of time (eg, at a temperature of 85 ° C to 90 ° C for 5 to 10 minutes). Essentially removed or reduced.

In some embodiments, esterase activity (eg, of any one esterase disclosed herein or at least disclosed herein) in the manner provided herein for producing a food product. At least one step in which the activity of any combination of the two esterases) is essentially eliminated or modulated is a recombinant microbial host cell capable of producing recombinant components and is essentially eliminated. Includes steps to obtain recombinant microbial host cells containing esterase activity that has been or modulated.

Thus, in some such embodiments, the methods provided herein can a) produce a recombinant component (eg, any of the recombinant components disclosed herein). Step to obtain a recombinant microbial host cell that is a recombinant microbial host cell and contains an esterase activity that is essentially eliminated or modulated relative to the esterase activity contained in the corresponding recombinant microbial host cell; b). Cultivate the recombinant microbial host cells in a culture medium under conditions suitable for the production and / or secretion of the recombinant component and / or the recombinant component to obtain a fermentation broth containing the recombinant component and essentially removed or modulated esterase activity. Steps; c) Purify the recombinant component from the fermentation broth as needed to obtain a preparation containing the recombinant component; and d) the recombinant component and essentially removed or modulated esterase activity. Includes steps to prepare a food product from a fermented broth or preparation containing.

Esterase activity is essentially the expression (ie, production of active protein) of an esterase (eg, any one esterase disclosed herein or any combination of at least two esterases disclosed herein). It can be essentially removed or modulated in recombinant microbial host cells by removing or modulating it, or by essentially removing or modulating the activity of the esterase. Essentially eliminating or modulating the expression of esterase in a recombinant microbial host cell is, for example, an esterase such that the expression of esterase is essentially eliminated or modulated in a recombinant microbial host cell. Control sequences that drive expression of (eg, promoter sequences, enhancer sequences, signal peptides, transcription termination factors, or any other sequence that controls gene expression (ie, transcription and / or translation)), or their function. (Ie, the part sufficient for the function of the control sequence) can be achieved by genetic modification.

Essentially eliminating or modulating the expression of an enzyme in a recombinant microbial host cell is, for example, an esterase such that the expression of an enzyme is essentially eliminated or modulated in a recombinant microbial host cell. A regulatory sequence that promotes the expression of the protein required for the expression of, or a functional portion thereof (eg, selected from transcription factors (eg, UniProt sequence G0RX49, G0RHG1, A0A2T4B416, A0A2T4BJU6, A2R2J1, A2R903, G2Q2Z5, and G2Q8I6). At least 50% identical to the sequence (eg, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more) Proteins containing amino acid sequences), post-translational modifying enzymes required for the production of active forms of esterase) Also achieved by: can do.

Essentially eliminating or modulating the expression of esterase in recombinant microbial host cells is, for example, complementary to, or specific to, esterase-encoding coding sequences in recombinant microbial host cells. It can also be achieved by introducing a recombinant polynucleotide containing a nucleotide sequence encoding an RNAi construct.

Essentially eliminating or modulating the expression or activity of esterase in a recombinant microbial host cell is, for example, a code encoding esterase or a functional portion thereof (eg, a catalytic domain) in a recombinant microbial host cell. It can also be achieved by genetically modifying the sequence.

Essentially eliminating or modulating the activity of an esterase in a recombinant microbial host cell may, for example, essentially eliminate or modulate the expression and / or activity of an endogenous inhibitor of esterase in the recombinant microbial host cell. By genetically modifying the regulatory sequence that promotes the expression of the endogenous inhibitor of esterase in recombinant microbial host cells and / or by genetically modifying the coding sequence that encodes the endogenous inhibitor of esterase so as to be modulated. , Or by introducing a recombinant polynucleotide encoding a heterologous inhibitor of esterase into a recombinant microbial host cell.

Genetic modification can occur, for example, by introducing, substituting, or removing one or more nucleotides in a nucleotide sequence. For example, inserting or removing one or more nucleotides to introduce a stop codon, removing the start codon, inserting a frame shift of an open reading frame, or a point mutation, a miss sense mutation, a substitution mutation, absent. Loss mutations, frame shift mutations, insertion mutations, duplicate mutations, amplification mutations, translocation mutations, or inversion mutations may be created.

Methods for genetically modifying microbial host cells are well known in the art and include random mutagenesis and screening, site-specific mutagenesis, PCR mutagenesis, insertion mutations, and chemical mutagenesis (eg, hydroxylamine, N). -Methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrite, ethylmethanesulfonic acid (EMS), sodium bisulfite, (Using formic acid, nucleotide analogs), irradiation (eg, UV (UV) irradiation), deletion of coding or non-coding nucleotide sequences, homologous recombination, FLP / FRT recombination, gene disruption, CRISPR gene editing, and Includes, without limitation, genetic conversion.

In some embodiments, esterase activity (eg, activity of any one esterase disclosed herein or disclosed herein) in the manner provided herein for producing food products. The activity of any combination of at least two esterases) is essentially eliminated or modulated by at least one step of purifying the recombinant component by separating from the esterase activity produced by the recombinant microbial host cell. include.

Thus, in some such embodiments, the methods provided herein are capable of producing a) recombinant components (eg, any of the recombinant components disclosed herein). Steps to obtain recombinant microbial host cells; b) Culturing the recombinant microbial host cells in a culture medium under conditions suitable for the production and / or secretion of the recombinant components to obtain a fermentation broth containing the recombinant components; c) Steps to purify the recombinant component from the fermentation broth to obtain a preparation containing the recombinant component so that the preparation contains essentially removed or modulated esterase activity; and d) the recombinant component. And comprises the step of preparing a food product from a preparation containing essentially eliminated or modulated esterase activity.

Purification of the recombinant component from the fermentation broth can be achieved based on any one property or combination of properties that distinguishes the recombinant component from esterase or is shared between the recombinant component and esterase. Non-limiting examples of such properties are size, molecular weight, degradation by a particular protease or combination of proteases, isoelectric point (pI), charge, thermal instability / thermal stability, affinity for a particular molecule, solubility. Includes pH stability, hydrophobicity, and hydrophilicity.

In some embodiments, the recombinant component is purified on the basis of thermal instability / thermal stability. In some such embodiments, fermented broths containing recombinant components (eg, clarified fermented broths (ie, fermented broths from which cells and cell debris have been removed)) or preparations have esterase denatured and settled from solution. However, the recombinant component is heated to a temperature that remains structurally complete and soluble. Precipitated esterases are subsequently various filtration techniques including, but not limited to, 1-g sedimentation, accelerated sedimentation by centrifugation, and / or depth filtration or tangential flow filtration using filter pads, sheets, or membranes. It can be separated from the soluble recombinant component by any suitable method, including.

In some embodiments, the recombinant component is purified on the basis of hydrophobicity. In some such embodiments, a suitable chromatograph carrier is added to the fermentation broth (eg, clarified fermentation broth) or preparation containing the recombinant component, and the pH and / or ion of the fermentation broth or preparation. The strength is adjusted so that the esterase or recombinant component (but not both) binds to the chromatograph carrier (eg, based on hydrophobic interactions), leaving the soluble recombinant or esterase in the unbound moiety, respectively. Will be done. Non-limiting examples of suitable chromatograph carriers include phenylcepharose, butylcepharose, and octylcepharose. Chromatograph carriers with bound esterase or recombinant components are subsequently made by any suitable method known in the art, including but not limited to 1-g precipitation, centrifugation, or filtration, respectively. It can be separated from soluble recombinant components or esterases. Alternatively, the chromatograph retainer can be a stationary retainer (eg, an adsorbent in a column) that transfers a fermentation broth or preparation containing the recombinant component, with the recombinant component obtained at the unbound moiety. The esterase is bound to the chromatograph carrier, or the recombinant component is bound to the chromatograph carrier, the esterase remains in the non-binding moiety, and the bound recombinant component is subsequently followed, for example, for example. It is released from the chromatograph carrier by adjusting the pH and / or ion intensity.

In some embodiments, the recombinant component is purified based on charge. In some such embodiments, a counterion or ion exchange resin or acid salt of sodium is added to the fermented broth (eg, clarified fermented broth) or preparation containing the recombinant component and the pH of the fermented broth or preparation. And / or ionic strength is adjusted so that the counterion or ionic exchange resin or sodium acid salt forms a complex with the recombinant or esterase, leaving the soluble esterase or recombinant component behind, respectively. Complexes subsequently include, but are not limited to, 1-g sedimentation, accelerated sedimentation by centrifugation, and / or depth filtration or tangential flow filtration using filter pads, sheets, or membranes. Can be isolated by any suitable method known in the art, including. In embodiments where the recombinant component is complexed with a counterion or ion exchange resin or a sodium acid salt, the recombinant component repels the recombinant component and the counterion or ion exchange resin or the acid salt of sodium. It can be extracted from the complex by adjusting the pH and / or ion intensity.

In some embodiments, the recombinant component is purified based on pH stability. In some such embodiments, the pH of the fermented broth (eg, clarified fermented broth) or preparation containing the recombinant component is such that the esterase is denatured and precipitates from the solution, leaving behind the soluble recombinant component. Is adjusted to. Precipitated esterases are subsequently subjected to a variety of filtration techniques including, but not limited to, 1-g sedimentation, accelerated sedimentation by centrifugation, and / or depth filtration or tangent flow filtration using filter pads, sheets, or membranes. It can be separated from the soluble recombinant component by any suitable method, including but not limited to these.

In some embodiments, the recombinant component is charged, thermally unstable / thermally stable, hydrophobic, size, molecular weight, pH stability, isoelectric point (pI), affinity for a particular molecule, and soluble. Purified on the basis of two or more of them, various strategies for separation are continuous (eg, charge-based separation followed by hydrophobic-based separation, hydrophobic-based separation followed by. Separation based on pH stability) or simultaneously (eg, separation based on pH stability and thermal instability / thermal stability, separation based on pH stability and affinity for a particular molecule, solubility and pH stability and / or heat. Instability / thermal stability and / or separation based on pI) is performed.

Esterase Activity The books provided herein that are essentially eliminated or modulated esterase activity (eg, contained in fermented broths or preparations containing recombinant components, or capable of producing recombinant components. The essentially eliminated or modulated esterase activity contained in the recombinant microbial host cells provided herein is or at least two (eg, at least two, at least three) of any one esterase. , At least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or more) of any combination of esterases. It is possible that the activity is essentially eliminated or modulated.

Non-limiting examples of such esterases include carboxyester hydrolase (EC number 3.1.1), phosphate diester hydrolase (EC number 3.1.4), exoribonuclease (EC number 3.1.13), and the like. Carboxylesterase (EC number 3.1.1.1), arylesterase (EC number 3.1.1.2), triacylglycerol lipase (EC number 3.1.1.3), phosphorlipase A2 (EC number 3) 1.1.1.4), lysophospholipase (EC number 3.1.1.5), acetylesterase (EC number 3.1.1.6), acetylcholinesterase (EC number 3.1.1.7), Phosphorlipase C (EC No. 3.1.4.3), Phosphorlipase D (EC No. 3.1.4.4), Phosphorinocitidophospholipase C (EC No. 3.1.4.11), Pectinesterase (EC No. 3.1.4.11) 3.1.1.11), gluconolactonase (EC number 3.1.1.17), acylglycerol lipase (EC number 3.1.1.23), 3-oxoadipate enollactonase (EC) No. 3.1.1.24), 1,4-lactonase (EC No. 3.1.1.25), galactolipase (EC No. 3.1.1.26), phosphoripase A1 (EC No. 3.1. 1.32), lipoprotein lipase (EC number 3.11.34), cephalosporin-C deacetylase (EC number 3.1.1.41), carboxymethylene buthenolidase (EC number 3.1. 1.45), 2-pyrone-4,6-dicarboxylic acid lactase (EC number 3.11.57), ferroyl esterase (EC number 3.11.73), cutinase (EC number 3.1) 1.74), and hormone-sensitive lipase (EC number 3.11.79).

Non-limiting examples of suitable carboxyl ester hydrolases include sequences selected from any of the sequences encoding carboxyl ester hydrolases listed in Table 1 and at least 50%, at least 55%, at least 60%, at least 65%. At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98 %, At least 99%, at least 99.5%, or 100% comprising an esterase comprising an amino acid sequence that is identical.

In some embodiments, the carboxyl ester hydrolases are UniProt sequences G0RIU1, A2R1N7, G0RBM4, A0A2T4BBP9, G2Q379, A0A2T4AZ21, A2R8Z3, G0R9X3, A2QPC2, G0RCG3, A2QPC2, G0RCG3, A2QPC2, G0RCG3, A0A2 At least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95 Includes an amino acid sequence having an amino acid sequence identity of%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%.

Non-limiting examples of suitable phosphate diester hydrolases include sequences selected from any of the sequences encoding phosphate diester hydrolases listed in Table 1 and at least 50%, at least 55%, at least 60%, at least 65. %, At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, It comprises an esterase containing an amino acid sequence that is at least 98%, at least 99%, at least 99.5%, or 100% identical.

Non-limiting examples of suitable exoribonucleases are at least 50%, at least 55%, at least 60%, at least 65%, at least 70 with sequences selected from any of the sequences encoding exoribonucleases listed in Table 1. %, At least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, Contains esterases comprising amino acid sequences that are at least 99%, at least 99.5%, or 100% identical.

Non-limiting examples of suitable carboxylesterases are at least 50%, at least 55%, at least 60%, at least 65%, at least 70 with sequences selected from any of the sequences encoding carboxylesterases listed in Table 1. %, At least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, It contains an esterase containing an amino acid sequence that is at least 99%, at least 99.5%, or 100% identical.

Non-limiting examples of suitable arylesterases are at least 50%, at least 55%, at least 60%, at least 65%, at least 70 with sequences selected from any of the sequences encoding arylesterases listed in Table 1. %, At least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, Includes esterases containing amino acid sequences that are at least 99%, at least 99.5%, or 100% identical.

Non-limiting examples of suitable triacylglycerol lipases are at least 50%, at least 55%, at least 60%, at least 65 with sequences selected from any of the sequences encoding triacylglycerol lipases listed in Table 1. %, At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, Contains esterases comprising amino acid sequences that are at least 98%, at least 99%, at least 99.5%, or 100% identical. In some embodiments, the triacylglycerol lipase is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80 with the sequence selected from the UniProt sequences A2QM14 and A2R709. %, At least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5. Includes an amino acid sequence having a% or 100% amino acid sequence identity.

Non-limiting examples of suitable phospholipase A2 are at least 50%, at least 55%, at least 60%, at least 65%, at least 70 with a sequence selected from any of the sequences encoding phospholipase A2 listed in Table 1. %, At least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, It comprises an esterase comprising an amino acid sequence having at least 99%, at least 99.5%, or 100% amino acid sequence identity.

Non-limiting examples of suitable lysophospholipases are at least 50%, at least 55%, at least 60%, at least 65%, at least 70 with sequences selected from any of the sequences encoding lysophospholipase listed in Table 1. %, At least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, It comprises an esterase containing an amino acid sequence that is at least 99%, at least 99.5%, or 100% identical. In some embodiments, the lysophospholipase is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75 with a sequence selected from the UniProt sequences G0RX90, G2Q0K1, A0A2T4B235, A2QC75 and A2QC42. %, At least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, Includes an amino acid sequence having at least 99.5% or 100% amino acid sequence identity.

Non-limiting examples of suitable acetylesterases are at least 50%, at least 55%, at least 60%, at least 65%, at least 70 with sequences selected from any of the sequences encoding acetylesterase listed in Table 1. %, At least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, It contains an esterase containing an amino acid sequence that is at least 99%, at least 99.5%, or 100% identical. In some embodiments, the acetylesterase is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80 with a sequence selected from the UniProt sequences G0RHJ4, A0A2T4BJD9 and G2QL32. %, At least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5. Includes an amino acid sequence having% or 100% amino acid sequence identity.

Non-limiting examples of suitable acetylcholinesterases are at least 50%, at least 55%, at least 60%, at least 65%, at least 70 with sequences selected from any of the sequences encoding acetylcholinesterase listed in Table 1. %, At least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, It comprises an esterase comprising an amino acid sequence that is at least 99%, at least 99.5%, or 100% identical.

Non-limiting examples of suitable phospholipase C are at least 50%, at least 55%, at least 60%, at least 65%, at least 70 with a sequence selected from any of the sequences encoding phospholipase C listed in Table 1. %, At least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, It comprises an esterase containing an amino acid sequence that is at least 99%, at least 99.5%, or 100% identical. In some embodiments, the phospholipase C is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80 with a sequence selected from the UniProt sequences G0REM9, A0A2T4BFY3 and A2QAD7. %, At least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5. Includes an amino acid sequence having a% or 100% amino acid sequence identity.

Non-limiting examples of suitable phospholipase D are at least 50%, at least 55%, at least 60%, at least 65%, at least 70 with sequences selected from any of the sequences encoding phospholipase D listed in Table 1. %, At least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, It comprises an esterase containing an amino acid sequence that is at least 99%, at least 99.5%, or 100% identical.

Non-limiting examples of suitable phosphoinositide phospholipase C are at least 50%, at least 55%, at least 60% of the sequences selected from any of the sequences encoding phosphoinositide phospholipase C listed in Table 1. At least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, At least 98%, at least 99%, at least 99.5%, or 100% comprising an esterase comprising an amino acid sequence that is identical.

Non-limiting examples of suitable pectinesterases are sequences selected from any of the sequences encoding pectinesterases listed in Table 1 and at least 50%, at least 55%, at least 60%, at least 65%, at least 70. %, At least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, It comprises an esterase comprising an amino acid sequence that is at least 99%, at least 99.5%, or 100% identical.

Non-limiting examples of suitable gluconolactonases are at least 50%, at least 55%, at least 60%, at least 65 with sequences selected from any of the sequences encoding gluconolactonase listed in Table 1. %, At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, It comprises an esterase containing an amino acid sequence that is at least 98%, at least 99%, at least 99.5%, or 100% identical.

Non-limiting examples of suitable acylglycerol lipases are at least 50%, at least 55%, at least 60%, at least 65%, with sequences selected from any of the sequences encoding acylglycerol lipases listed in Table 1. At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98 %, At least 99%, at least 99.5%, or 100% comprising an esterase comprising an amino acid sequence that is identical.

Non-limiting examples of suitable 3-oxoadipate enol lactonases are at least 50%, at least 55, with sequences selected from any of the sequences encoding 3-oxoadipate enol lactonase listed in Table 1. %, At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, It comprises an esterase comprising an amino acid sequence that is at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical.

Non-limiting examples of suitable 1,4-lactonases are at least 50%, at least 55%, at least 60%, with sequences selected from any of the sequences encoding 1,4-lactonase listed in Table 1. At least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, At least 98%, at least 99%, at least 99.5%, or 100% comprising an esterase comprising an amino acid sequence that is identical.

Non-limiting examples of suitable galactolipases are at least 50%, at least 55%, at least 60%, at least 65%, at least 70 with sequences selected from any of the sequences encoding galactolipase listed in Table 1. %, At least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, Contains esterases containing amino acid sequences that are at least 99%, at least 99.5%, or 100% identical.

Non-limiting examples of suitable phospholipase A1 are at least 50%, at least 55%, at least 60%, at least 65%, at least 70 with sequences selected from any of the sequences encoding phospholipase A1 listed in Table 1. %, At least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, It comprises an esterase containing an amino acid sequence that is at least 99%, at least 99.5%, or 100% identical.

Non-limiting examples of suitable lipoprotein lipases are at least 50%, at least 55%, at least 60%, at least 65%, with sequences selected from any of the sequences encoding lipoprotein lipase listed in Table 1. At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98 %, At least 99%, at least 99.5%, or 100% comprising an esterase comprising an amino acid sequence that is identical.

Non-limiting examples of suitable cephalosporin-C deacetylases are at least 50%, at least 55%, at least 60 of the sequences selected from any of the sequences encoding cephalosporin-C deacetylases listed in Table 1. %, At least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Includes esterases containing amino acid sequences that are at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical.

Non-limiting examples of suitable carboxymethylenebutenolidases include at least 50%, at least 55%, at least 60% of the sequences selected from any of the sequences encoding carboxymethylenebutenolidase listed in Table 1. At least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97 %, At least 98%, at least 99%, at least 99.5%, or 100% comprising an esterase comprising an amino acid sequence that is identical.

Non-limiting examples of suitable 2-pyrone-4,6-dicarboxylic acid lactonases include sequences selected from any of the sequences encoding 2-pyrone-4,6-dicarboxylic acid lactonase listed in Table 1. At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94 %, At least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% comprising an esterase comprising an amino acid sequence that is identical.

Non-limiting examples of suitable ferroyl esterases are at least 50%, at least 55%, at least 60%, at least 65%, with sequences selected from any of the sequences encoding ferroyl esterase listed in Table 1. At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98 %, At least 99%, at least 99.5%, or 100% comprising an esterase comprising an amino acid sequence that is identical.

Non-limiting examples of suitable cutinase are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, with sequences selected from any of the sequences encoding cutinase listed in Table 1. At least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 %, At least 99.5%, or 100% comprising an esterase comprising an amino acid sequence that is identical. In some embodiments, the cutinase is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least with the sequence selected from the UniProt sequences G0RH85, A2QBP1, A0A2T4BJB5, A5ABE6, A2R2W3 and G2QH51. 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , Includes an amino acid sequence having at least 99.5%, or 100% amino acid sequence identity.

Non-limiting examples of suitable hormone-sensitive lipases are at least 50%, at least 55%, at least 60%, at least 65%, with sequences selected from any of the sequences encoding hormone-sensitive lipases listed in Table 1. At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98 %, At least 99%, at least 99.5%, or 100% comprising an esterase comprising an amino acid sequence that is identical.

Non-limiting examples of additional suitable esterases are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85 with the sequences listed in Table 1. %, At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100 % Esterases containing amino acid sequences that are identical, and at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least in any one of the InterPro domains listed in Table 2. 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99. Includes esterases comprising an amino acid sequence having 5% or 100% amino acid sequence identity.

Esterases disclosed herein include esterases, which are conservatively modified variants of the esterases disclosed herein. As used herein, the term "conservatively modified variant" refers to an individual substitution, deletion, or addition to the encoded amino acid sequence, whereby the amino acids are chemically similar. Is replaced with an amino acid. Conservative substitution tables that provide functionally similar amino acids are well known in the art. The esterases disclosed herein further include polymorph variants, interspecific homologues, and alleles of the esterases disclosed herein.

Recombinant microorganisms provided herein that are essentially eliminated or modulated with esterase activity (eg, contained in fermented broths or preparations containing recombinant components or capable of producing recombinant components). In embodiments where the essentially eliminated or modulated esterase activity contained in the host cell contains the essentially eliminated or modulated activity of at least two esterases, at least two such esterases. Esterases may be one or more (eg, two or more, three or more, four or more, five or more, six or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, etc.) Any esterase of (eg, any esterase provided herein) can be included.

Non-limiting examples of such esterases include carboxyl ester hydrolase (EC number 3.1.1), phosphate diester hydrolase (EC number 3.1.4), exoribonuclease (EC number 3.1.13), and the like. Carboxylesterase (EC number 3.1.1.1), arylesterase (EC number 3.1.1.2), triacylglycerol lipase (EC number 3.1.1.3), phosphorlipase A2 (EC number 3) 1.1.1.4), lysophospholipase (EC number 3.1.1.5), acetylesterase (EC number 3.1.1.6), acetylcholinesterase (EC number 3.1.1.7), Phosphorlipase C (EC No. 3.1.4.3), Phosphorlipase D (EC No. 3.1.4.4), Phosphorinocitidophospholipase C (EC No. 3.1.4.11), Pectinesterase (EC No. 3.1.4.11) 3.1.1.11), gluconolactonase (EC number 3.1.1.17), acylglycerol lipase (EC number 3.1.1.23), 3-oxoadipate enollactonase (EC) No. 3.1.1.24), 1,4-lactonase (EC No. 3.1.1.25), galactolipase (EC No. 3.1.1.26), phosphoripase A1 (EC No. 3.1. 1.32), lipoprotein lipase (EC number 3.11.34), cephalosporin-C deacetylase (EC number 3.1.1.41), carboxymethylene buthenolidase (EC number 3.1. 1.45), 2-pyrone-4,6-dicarboxylic acid lactase (EC number 3.11.57), ferroyl esterase (EC number 3.11.73), cutinase (EC number 3.1) 1.74), hormone-sensitive lipase (EC number 3.11.79), and combinations thereof.

Non-limiting examples of such combinations are: one or more cutinase and one or more other carboxyl ester hydrolases; one or more lysophospholipases and one or more other carboxyl ester hydrolases; One or more triacylglycerol lipases and one or more other carboxyl ester hydrolases; one or more phosphorlipases A2 and one or more other carboxyl ester hydrolases; one or more phosphorlipases C and one Or multiple other carboxyl ester hydrolases; one or more acetylxylan esterases and one or more other carboxyl ester hydrolases; one or more extracellular lipase-like proteins and one or more other carboxyl ester hydrolases. One or more acetylesterases and one or more other carboxyl ester hydrolases; one or more GDSL lipases and one or more other carboxyl ester hydrolases; one or more alpha / beta hydrolase and 1 One or more other carboxyl ester hydrolases; one or more transcription factors that regulate the expression of esterase and one or more other carboxyl ester hydrolases; one or more cutinase and one or more lysophospholipases; One or more cutinase and one or more triacylglycerol lipases; one or more cutinase and one or more phosphorlipases A2; one or more cutinase and one or more phosphorlipases C; one or more Multiple cutinase and one or more acetylxylan esterases; one or more cutinase and one or more extracellular lipase-like proteins; one or more cutinase and one or more acetylesterases; one or more Cutinase and one or more GDSL lipases; one or more cutinase and one or more alpha / beta hydrolases; one or more transcription factors that regulate the expression of one or more cutinase and esterase; 1 One or more lysophospholipases and one or more triacylglycerol lipases; one or more lysophospholipases and one or more Phospholipase A2; one or more lysophospholipases and one or more phospholipases C; one or more lysophospholipases and one or more acetyloxylanesterases; one or more lysophospholipases and one or more Extracellular lipase-like protein; one or more lysophospholipases and one or more acetylesterases; one or more lysophospholipases and one or more GDSL lipases; one or more lysophospholipases and one or more Alpha / beta hydrolase; one or more transcription factors that regulate the expression of one or more lysophospholipases and esterases; one or more triacylglycerol lipases and one or more phospholipases A2; one or more Triacylglycerol lipase and one or more phospholipases C; one or more triacylglycerol lipases and one or more acetylxylan esterases; one or more triacylglycerol lipases and one or more extracellular Lipase-like proteins; one or more triacylglycerol lipases and one or more acetylesterases; one or more triacylglycerol lipases and one or more GDSL lipases; one or more triacylglycerol lipases and One or more alpha / beta hydrolases; one or more transcription factors that regulate the expression of one or more triacylglycerol lipases and esterases; one or more phospholipases A2 and one or more phospholipases C; One or more phospholipase A2 and one or more acetyloxylanesterases; one or more phospholipase A2 and one or more extracellular lipase-like proteins; one or more phospholipase A2 and one or more acetyls Estelase; one or more phospholipase A2 and one or more GDSL lipases; one or more phospholipase A2 and one or more alpha / beta hydrolase; regulates the expression of one or more phospholipase A2 and esterase One or more transcription factors; one or more phospholipase Cs and one or more acetyloxylanestera -Seses; one or more phospholipase Cs and one or more extracellular lipase-like proteins; one or more phospholipase Cs and one or more acetylesterases; one or more phospholipase Cs and one or more GDSL lipase; one or more phospholipase C and one or more alpha / beta hydrolase; one or more transcription factors that regulate the expression of one or more phospholipase C and esterase; one or more acetyls Xysilaneesterase and one or more extracellular lipase-like proteins; one or more acetylxylan esterases and one or more acetylesterases; one or more acetylxylan esterases and one or more GDSL lipases; one Or multiple acetylxylan esterases and one or more alpha / beta hydrolase; one or more transcription factors that regulate the expression of one or more acetylxylan esterases and esterases; one or more acetylesterases and one. Or multiple GDSL lipases; one or more acetylesterases and one or more alpha / beta hydrolase; one or more transcription factors that regulate the expression of one or more acetylesterases and esterases; one or more GDSL lipase and one or more alpha / beta hydrolases; one or more transcription factors that regulate the expression of one or more GDSL lipase and esterase; expression of one or more alpha / beta hydrolase and esterase. Regulating one or more transcription factors; one or more cutinase and one or more lysophospholipases and one or more other carboxyl ester hydrolases; one or more cutinase and one or more triacyl Gglycerol lipase and one or more other carboxyl ester hydrolases; one or more cutinase and one or more phospholipase A2 and one or more other carboxyl ester hydrolases; one or more cutinase and one or more Multiple phospholipase Cs and one or more other carboxyl ester hydrolases; one or more cutinase and one or more acetylxylan es Terase and one or more other carboxyl ester hydrolases; one or more cutinase and one or more extracellular lipase-like proteins and one or more other carboxyl ester hydrolases; one or more cutinase and 1 One or more acetylesterases and one or more other carboxyl ester hydrolases; one or more cutinase and one or more GDSL lipases and one or more other carboxyl ester hydrolases; one or more cutinase And one or more alpha / beta hydrolases and one or more other carboxyl ester hydrolases; one or more transcription factors and one or more other that regulate the expression of one or more cutinase and esterase. Carboxyl ester hydrolase; one or more lysophospholipases and one or more triacylglycerol lipases and one or more other carboxyl ester hydrolases; one or more lysophospholipases and one or more phosphorlipases A2 and 1 One or more other carboxyl ester hydrolases; one or more lysophospholipases and one or more phosphorlipases C and one or more other carboxyl ester hydrolases; one or more lysophospholipases and one or more Acetylxylanesterase and one or more other carboxyl ester hydrolases; one or more lysophospholipases and one or more extracellular lipase-like proteins; one or more lysophospholipases and one or more acetylesterases and One or more other carboxyl ester hydrolases; one or more lysophospholipases and one or more GDSL lipases and one or more other carboxyl ester hydrolases; one or more lysophospholipases and one or more Alpha / beta hydrolase and one or more other carboxyl ester hydrolases; one or more transcription factors and one or more other carboxyl ester hydrolases that regulate the expression of one or more lysophospholipases and esterases; One or more triacylglycerol lipases and one or more Multiple phosphoripase A2 and one or more other carboxyl ester hydrolases; one or more triacylglycerol lipases and one or more phosphorlipase C and one or more other carboxyl ester hydrolases; one or more Triacylglycerol lipase and one or more acetylxylan esterases and one or more other carboxyl ester hydrolases; one or more triacylglycerol lipases and one or more extracellular lipase-like proteins and one or more Other carboxyl ester hydrolases; one or more triacylglycerol lipases and one or more acetylesterases and one or more other carboxylester hydrolases; one or more triacylglycerol lipases and one or more GDSL lipase and one or more other carboxyl ester hydrolases; one or more triacylglycerol lipases and one or more alpha / beta hydrolases and one or more other carboxyl ester hydrolases; one or more One or more transcription factors and one or more other carboxyl ester hydrolases that regulate the expression of triacylglycerol lipases and esterases; one or more phosphorlipases A2 and one or more phosphorlipases C and one or more. Multiple other carboxyl ester hydrolases; one or more phosphorlipase A2 and one or more acetylxylan esterases and one or more other carboxyl ester hydrolases; one or more phosphorlipase A2 and one or more cells External lipase-like protein and one or more other carboxyl ester hydrolases; one or more phosphorlipase A2 and one or more acetylesterases and one or more other carboxyl ester hydrolases; one or more phosphorlipase A2 And one or more GDSL lipases and one or more other carboxyl ester hydrolases; one or more phosphorlipases A2 and one or more alpha / beta hydrolases and one or more other carboxyl ester hydrolases; 1 One or more hosts One or more transcription factors and one or more other carboxyl ester hydrolases that regulate the expression of holipase A2 and esterase; one or more phosphorlipase C and one or more acetylxylan esterases and one or more Other carboxyl ester hydrolases; one or more phosphoripase C and one or more extracellular lipase-like proteins; one or more phosphorlipase C and one or more acetylesterases and one or more other carboxyl esters Hydrolase; one or more phosphoripase C and one or more GDSL lipase and one or more other carboxyl ester hydrolases; one or more phosphorlipase C and one or more alpha / beta hydrolase and one or more Multiple other carboxyl ester hydrolases; one or more transcription factors that regulate the expression of one or more phosphorlipase C and esterases and one or more other carboxyl ester hydrolases; one or more acetylxylan esterases and One or more extracellular lipase-like proteins and one or more other carboxyl ester hydrolases; one or more acetylxylan esterases and one or more acetylesterases and one or more other carboxyl ester hydrolases; One or more acetylxylan esterases and one or more GDSL lipases and one or more other carboxyl ester hydrolases; one or more acetylxylan esterases and one or more alpha / beta hydrolase and one or more Multiple other carboxyl ester hydrolases; one or more transcription factors that regulate the expression of one or more acetylxylan esterases and esterases and one or more other carboxyl ester hydrolases; one or more acetylesterases and One or more GDSL lipases and one or more other carboxyl ester hydrolases; one or more acetylesterases and one or more alpha / beta hydrolases and one or more other carboxyl ester hydrolases; one Or multiple acetylesterases and esterers One or more transcription factors and one or more other carboxyl ester hydrolases that regulate the expression of ze; one or more GDSL phospholipases and one or more alpha / beta hydrolases and one or more other Carboxyl ester hydrolase; one or more transcription factors that regulate the expression of one or more GDSL phospholipases and esterases and one or more other carboxyl ester hydrolases; expression of one or more alpha / beta hydrolase and esterase. One or more transcription factors and one or more other carboxyl ester hydrolases; one or more cutinase and one or more lysophospholipases and one or more triacylglycerol lipases; one or more Multiple cutinase and one or more lysophospholipases and one or more phospholipases A2; one or more cutinase and one or more lysophospholipases and one or more phospholipases; one or more cutinase and one One or more lysophospholipases and one or more acetylxylan esterases; one or more cutinase and one or more lysophospholipases and one or more extracellular lipase-like proteins; one or more cutinase and one One or more lysophospholipases and one or more acetylesterases; one or more cutinase and one or more lysophospholipases and one or more GDSL lipases; one or more cutinase and one or more Resophospholipase and one or more alpha / beta hydrolase; one or more cutinase and one or more transcription factors that regulate the expression of one or more lysophospholipase and esterase; one or more cutinase and one One or more triacylglycerol lipases and one or more phospholipases A2; one or more cutinase and one or more triacylglycerol lipases and one or more phospholipases C; one or more cutinase and 1 One or more triacylglycerol phospholipases and one or more acetylxylan esterases; one or more cutinase and one or more totes Riacylglycerol lipase and one or more extracellular lipase-like proteins; one or more cutinase and one or more triacylglycerol lipase and one or more acetylesterases; one or more cutinase and one Or multiple triacylglycerol lipases and one or more GDSL lipases; one or more cutinase and one or more triacylglycerol lipases and one or more alpha / beta hydrolase; one or more cutinase and One or more transcription factors that regulate the expression of one or more triacylglycerol lipases and esterases; one or more cutinase and one or more phospholipases A2 and one or more phospholipases C; one or more Multiple cutinase and one or more phospholipase A2 and one or more acetylxylan esterase; one or more cutinase and one or more phospholipase A2 and one or more extracellular lipase-like proteins; one or more Multiple cutinase and one or more phospholipase A2 and one or more acetylesterases; one or more cutinase and one or more phospholipase A2 and one or more GDSL lipase; one or more cutinase and One or more phospholipase A2 and one or more alpha / beta hydrolase; one or more cutinase and one or more transcription factors that regulate the expression of one or more phospholipase A2 and esterase; one or more Multiple cutinase and one or more phospholipase C and one or more acetylxylan esterase; one or more cutinase and one or more phospholipase C and one or more extracellular lipase-like proteins; one or more Multiple cutinase and one or more phospholipase C and one or more acetylesterases; one or more cutinase and one or more phospholipase C and one or more GDSL lipase; one or more cutinase and One or more phospholipase C and one or more alpha / beta hydrolase; one or more cutinase and one or more phosphory One or more transcription factors that regulate the expression of parse C and esterase; one or more cutinase and one or more acetylxylan esterase and one or more extracellular lipase-like proteins; one or more cutinase And one or more acetylxylan esterases and one or more acetylesterases; one or more cutinase and one or more acetylxylan esterases and one or more GDSL lipases; one or more cutinase and one One or more acetylxylan esterases and one or more alpha / beta hydrolase; one or more cutinase and one or more transcription factors that regulate the expression of one or more acetylxylan esterases and esterases; one Or multiple cutinase and one or more extracellular lipase-like proteins and one or more acetylesterase; one or more cutinase and one or more extracellular lipase-like proteins and one or more GDSL lipase; Expression of one or more cutinase and one or more extracellular lipase-like proteins and one or more alpha / beta hydrolase; one or more cutinase and one or more extracellular lipase-like proteins and esterase Regulating one or more transcription factors; one or more cutinase and one or more acetylesterase and one or more GDSL lipases; one or more cutinase and one or more acetylesterase and one Or multiple alpha / beta hydrolases; one or more cutinase and one or more transcription factors that regulate the expression of one or more acetylesterases and esterases; one or more cutinase and one or more GDSL Lipase and one or more alpha / beta hydrolase; one or more cutinase and one or more transcription factors that regulate the expression of one or more GDSL lipase and esterase; and one or more cutinase and one Contains one or more alpha / beta hydrolase fold domain-containing proteins and one or more transcription factors that regulate the expression of esterase.

Producing a recombinant ingredient in at least one step of the method provided herein for producing a food product, or in a fermented broth or preparation provided herein comprising a recombinant ingredient. Appropriate numbers and / or combinations of esterases whose activity must be essentially eliminated or modulated in the recombinant microbial host cells provided herein are specified by methods known in the art. Can be done. For example, esterases are isolated by methods known in the art (eg, affinity chromatography, zymogram assay, gel electrophoresis) and tested in vitro to either one or either of at least two esterases. Can be determined whether the combination of the above provides a significant amount of degradation of a particular diglyceride, triglyceride, phospholipid, or lipoprotein. Moreover, recombinant components (eg, any recombinant component disclosed herein) can be produced and are essentially eliminated in any one or any combination of at least two esterases or Recombinant microbial host cells with modulated activity can be obtained, and the degree of reduction or elimination of lipid degradation in food products containing recombinant components produced by each such recombinant microbial host cell is of this degree. It can be measured by a method known in the art.

In some embodiments, the fermented broths or preparations provided herein that contain recombinant components, or the recombinant microbial host cells provided herein that are capable of producing recombinant components, are specific. Includes esterase activity combinations and / or levels that provide a specific profile of esterase activity (i.e., esterase activity profile) useful in the production of food products. In some such embodiments, the esterase activity profile is optimized to provide the desired flavor, aroma, texture, emulsification, nutritional content, and / or shelf life of the food product.

Esterase activity can be measured using an enzyme assay. For example, the enzyme activity can be detected using a colorimetric reaction product or, for example, a PAGE gel, a spectrophotometer, imaging, UV / Vis, light, and HPLC (eg, a triglyceride esterase). It can be determined by the production of free fatty acids and / or glycerol) produced from catalytic hydrolysis.

Recombinant Microbial Host Cell Producing Recombinant Component and Containing Essentially Removed or Modulated Esterase Activity In another embodiment, the recombinant component can be produced and contained in the corresponding recombinant microbial host cell. Esterase activity that is essentially eliminated or modulated as compared to the esterase activity (eg, the activity of any one esterase disclosed herein or at least two esterases disclosed herein). Recombinant microbial host cells comprising any combination of activity) are provided herein.

The esterase activity contained in the recombinant microbial host cells provided herein can be essentially eliminated or modulated by any means. In some embodiments, esterase activity essentially eliminates or modulates esterase expression (ie, production of an active protein), or essentially eliminates or modulates esterase activity. Is essentially removed or modulated by. Thus, in various embodiments, the recombinant microbial host cells provided herein are a) a regulatory sequence that promotes the expression of esterase, or a functional portion thereof (ie, a portion sufficient for the function of the regulatory sequence). ) (For example, a promoter sequence, an enhancer sequence, a signal peptide, a transcription termination factor, or any other sequence that controls the expression (ie, transcription and / or translation) of a gene) (where the genetic modification is an esterase). (Here, the genetic modification essentially eliminates or modulates the activity of the esterase); b) a genetic modification of the coding sequence encoding the esterase or its functional portion (eg, the catalytic domain). Remove or modulate); c) A regulatory sequence that promotes the expression of proteins required for the expression of esterase (eg, transcription factors, post-translational modifying enzymes required for the production of active forms of esterase), or functional portions thereof. Genetic modification of (ie, the portion sufficient for the function of the regulatory sequence) (where the genetic modification essentially eliminates or modulates the expression of the protein required for the expression of esterase, thereby essence the expression of esterase. (Eg); d) Genetic modification of the coding sequence encoding the protein or functional portion thereof (eg, the DNA binding domain of the transcription factor, the catalytic domain of the post-translation modification enzyme) required for the expression of esterase. (Here, genetic modification essentially eliminates or modulates the activity of the protein required for esterase expression, thereby essentially eliminating or modulating the expression of esterase); e) Intrinsic of esterase. Genetic modification of the regulatory sequence that promotes the expression of the inhibitor, or its functional portion (ie, sufficient portion for the function of the regulatory sequence) (where the genetic modification essentially eliminates or eliminates the expression of the endogenous inhibitor. Modulate and thereby essentially eliminate or modulate the expression of esterase); f) Genetic modification of the coding sequence encoding the endogenous inhibitor of esterase (where the genetic modification is the activity of the endogenous inhibitor). Essentially eliminates or modulates, thereby essentially eliminating or modulating the expression of esterase); and / or g) a coding sequence encoding a heterologous (ie, non-natural) inhibitor of esterase. Introduced Includes genetic modification (where, genetic modification provides the production of heterologous inhibitors, thereby essentially eliminating or modulating the expression of esterase).

The genetic modification can consist, for example, the insertion, substitution, or deletion of one or more nucleotides in the polynucleotide sequence. Genetic modifications, for example, introduce stop codons; move start codons; insert frame shifts in open reading frames; or point mutations, missense mutations, substitution mutations, deletion mutations, frame shift mutations. , Insert mutations, duplicate mutations, proliferation mutations, translocation mutations, or inversion mutations can be created.

In some embodiments, the recombinant microbial host cells provided herein are esterases (eg, any one esterase disclosed herein or at least two esterases disclosed herein. Includes recombinant polynucleotides containing nucleotide sequences that are complementary to the coding sequence encoding (any combination), encode an RNAi construct specific for esterase, or encode a heterologous inhibitor of esterase activity.

In some embodiments, the recombinant microbial host cells provided herein include genetic modification that essentially eliminates or modulates the expression or activity of a protein required for expression of esterase. The required such protein is a transcription factor that regulates the expression of esterase. In some such embodiments, the transcription factor that regulates the expression of esterase is at least 50% with a sequence selected from any of the sequences encoding the transcription factors that regulate the expression of esterase listed in Table 1. At least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least Includes an amino acid sequence having 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% amino acid sequence identity. In some such embodiments, the transcription factors that regulate the expression of esterase are at least 50%, at least 50% of the sequences selected from the UniProt sequences G0RX49, G0RHG1, A0A2T4B416, A0A2T4BJU6, A2R2J1, A2R903, G2Q2Z5, and G2Q8I6. %, At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, Includes an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% amino acid sequence identity. In some such embodiments, the recombinant microbial host cells provided herein essentially exhibit or act on the expression or activity of a combination of at least two transcription factors that regulate the expression of one or more esterases. Includes one or more genetic modifications to be removed or modulated.

The recombinant microbial host cells provided herein are capable of producing recombinant components and are at least 10%, at least 15%, at least 20% of the esterase activity contained in the corresponding recombinant microbial host cells. %, At least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, Esterase activity reduced by at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (eg, any one disclosed herein). The activity of one esterase or any combination of at least two esterases disclosed herein). In some such embodiments, the recombinant microbial host cells provided herein contain essentially eliminated esterase activity. In other embodiments, the recombinant microbial host cells provided herein are capable of producing recombinant components and are at least 25%, at least, at least 25% of the esterase activity contained in the corresponding recombinant microbial host cells. 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1 Includes an esterase activity that is increased by 000% (eg, the activity of any one esterase disclosed herein or the activity of any combination of at least two esterases disclosed herein).

Microbial Host Cell The recombinant microbial host cell provided herein can be derived from any microbial cell. In some embodiments, the recombinant microbial host cell provided herein is a recombinant bacterial host cell. Recombinant bacterial host cells can be derived from any bacterial strain known in the art. Non-limiting examples of bacterial strains are Bacillus phylum, Cyanobacterium (Orchid algae), Oscillatriophcideae, Bacillus, Lactobacillus, Euremo, Bacillus, Lactobacillus, Acetbactor suboxydance. (Acetobacter suboxydans), Acetobacter xylinum, Actinoplanes misouriensis, Artrosspira platensis (Arthrospila platensis) spiras Bacillus coagulans, Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, Bacillus licheniformis, Bacillus stealothermophilus (Escherichia coli), Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactococcus lactis, Lactococcus lactis, Lactococcus lactis, Lactococcus lactis. Lactobacillus reuteri, Leuconostock citrovorum, Leuconostoc dextranicum, Leuconostock dextranicum, Leuconostoc mesenteros Micrococcus lysodeictus, Spirulina, Streptococcus cremoris, Streh Putokokkasu lactis (Streptococcus lactis), Streptococcus lactis subspecies Jiasechirakuchisu (Streptococcus lactis subspecies diacetylactis), Streptococcus thermophilus (Streptococcus thermophilus), Streptomyces chatter Noo thuringiensis (Streptomyces chattanoogensis), Streptomyces griseus (Streptomyces Griseus, Streptomyces natalensis, Streptomyces olivaseus, Streptomyces olivochromenes, Streptomyces olivochrome Tetrahymena thermophile), Tetrahymena Hegewisuki (Tetrahymena hegewischi), Tetrahymena Hiperangurarisu (Tetrahymena hyperangularis), Tetrahymena Marasenshisu (Tetrahymena malaccensis), Tetrahymena pigment over support (Tetrahymena pigmentosa), Tetrahymena pyriformis (Tetrahymena pyriformis), and Tetrahymena borax (Tetrahymena vorax), Xanthomonas campestris, and their derivatives and hybrids.

In some embodiments, the recombinant microbial host cell provided herein is a recombinant yeast host cell. Recombinant yeast host cells can be derived from any yeast strain known in the art. Non-limiting examples of yeast strains include Candida albicans, Candida etchellsii, Candida gilliermondii, Candida pichia, Candida pichia, and Candida pichia. ), Candida shoe de tropicalis (Candida pseudotropicalis), torula yeast (Candida utilis), Candida Basachirisu (Candida versatilis), Debaryomyces-Hanzeni (Debaryomyces hansenii), Eremoteshiumu Ashby (Eremothecium ashbyii), Hansenula polymorpha (Hansenula polymorpha) , Kluyveromyces sp., Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces pyrace ), Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membrane facien (Pichia mi) Ogatea minuta, Pichia lindneri, Pichia opuntiae, Pichia thermotrelans, Pichia thericatelia, Pichia salictaria Pichia pijperi), Pichia stippis, Pichia methanolica (P) ichia methanolica), Rhodotorula sp. ), Saccharomyces species (Saccharomyces sp.), Saccharomyces bayanus (Saccharomyces bayanus), Saccharomyces Betikasu (Saccharomyces beticus), budding yeast (Saccharomyces cerevisiae), Saccharomyces Chebarieri (Saccharomyces chevalieri), Saccharomyces Deer statics (Saccharomyces diastaticus) , Saccharomyces ellipsometry Lee Deus (Saccharomyces ellipsoideus), Saccharomyces Igujigusu (Saccharomyces exiguus), Saccharomyces Furorenchinusu (Saccharomyces florentinus), Saccharomyces fragilis (Saccharomyces fragilis), brewer's yeast (Saccharomyces pastorianus), fission yeast (Saccharomyces pombe), sake Yeast (Saccharomyces sake), Saccharomyces uvalum, Saccharomyces roseus, Saccharomyces roseus, Saccharomyces saccharomyces, Saccharomyces saccharomyces, Saccharomyces, Saccharomyces, Saccharomyces, Saccharomyces ..

In some embodiments, the recombinant microbial host cell provided herein is a recombinant filamentous fungal host cell. Recombinant filamentous fungus host cells can be derived from any filamentous strain known in the art, including any holomorphic, telemorphic, or anamorphic form thereof. Non-limiting examples of filamentous strains include the genus Acremonium, the genus Aspergillus, the genus Aureobasideium, the genus CANariomyces, the genus Chaetonium, the genus Ketomium. Corynascus, Cryptococcus, Chrysosporium, Coonemeria, Dactylomyces, Emerisilia, Emericella, Fusarium, Gibbella, Humicola, Lentinula, Magnaporte, Malbranchium, Melanocarpus, Melanocarpus, Melanocarpus Genus Myceliophsora, Genus Myrothecium, Genus Neocalimastix, Genus Neurospora, Genus Pecilomyces, Genus Pecilomyces, Genus Penicylium ), Piromyces, Rhizopus, Schizophyllum, Cytalidium, Sporotichum, Sterotrichum, Sterotrichum, Stereoth ), Thermomyces, Thielavia, Polypocladium, and Trichoderma strains.

Non-limiting examples of Acremonium strains include Acremonium alabamense. Non-limiting examples of Aspergillus strains include Aspergillus acleatus, Aspergillus awamori, Aspergillus clavatus, Aspergillus aspergillus, Aspergillus aspergillus.・ Aspergillus fumigatus, Aspergillus japonix, Aspergillus nidulans, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus aspergillus. Aspergillus sojae, and Aspergillus terreus, as well as Emericella, Neosartora, and Petromyces spes. Non-limiting examples of Chrysosporium genus strains include Chrysosporium botryoides, Chrysosporium carmichaeri, Chrysosporium chrysosporium, Chrysosporium, Chrysosporium, Chrysosporium, Chrysosporium, Chrysosporium, Chrysosporium, and Chrysosporium. Chrysosporium europae), Chrysosporium Eborusean'nui (Chrysosporium evolceannui), Chrysosporium Farinikora (Chrysosporium farinicola), Chrysosporium Fasuchijiumu (Chrysosporium fastidium), Chrysosporium Firihorume (Chrysosporium filiforme), Chrysosporium Georugie ( Chrysosporium guergiae, Chrysosporium globiferum, Chrysosporium globiferium variant Chrysosporium globiferum vari. Chrysosporium hirundo, Chrysosporium hispanicum, Chrysosporium holmi, Chrysosporium chrysosporium, Chrysosporium chrysosporium, Chrysosporium chrysosporium Chrysosporium chrysosporium, Chrysosporium chrysosporium, Chrysosporium chrysosporium, Chrysosporium kuzurovianum, Chrysosporium chrysosporium Chrysosporium lucnowense, Chrysosporium lucknowwense Garg 27K, Chrysosporium medium, Chrysosporium varieties, Chrysosporium medium, Chrysosporium medium, Chrysosporium medium. chrysosporium chrysosporium mephrysporium chrysosporium merdarium, chrysosporium merdarium variant roseum (Chrysosporium minor) chrysosporium minor chrysosporium chrysosporium Chrysosporium pannicola, Chrysosporium parvum, Chrysosporium parvum variant cressens (Chrysosporium parvum var. Chrysosporium chrysosporium chrysosporium chrysosporium chrysosporium chrysosporium chrysosporium chrysosporium chrysosporium chrysosporium chrysosporium chrysosporium chrysosporium chrysosporium chrysosporium chrysosporium chrysosporium chrysosporium chrysosporium chrysosporium chrysosporium chrysosporium Chrysosporium synchronum, Chrysosporium tropicum, Chrysosporium andratum, Chrysosporium undulutum, Chrysosporium valrens , And Chrysosporium zonatum.

Non-limiting examples of Fusarium genus strains include Fusarium moniliform, Fusarium venenatum, Fusarium oxysporum, Fusarium glaminearum, Fusarium glaminearum, Fusarium glaminearum. , Fusarium verticiollioides, Fusarium mulmorum, Fusarium clookwellense, Fusarium foliaus poire Includes Fusarium sambuccinum, and Fusarium turulosum, as well as their associated diberella telemorphic forms. Non-limiting examples of strains of the genus Myceriophthora include Myceriophthora thermophila. Non-limiting examples of strains of the genus Mucor are Mucor miehei var. Cooney et Emerson (Rhizomucor miehei) (Cooney & R. ) Includes Lindt. Non-limiting examples of strains of the genus Neurospora include Neurospora crassa.

Non-limiting examples of Penicillium genus strains include Penicillium chrysogenum and Penicillium roquefortii. Non-limiting examples of strains of the genus Rhizopus include Rhizopus niveus. Non-limiting examples of strains of the genus Sporoticum include Sporoticum cellulophyllum. Non-limiting examples of strains of the genus Thieravia include Thielavia terrestris. Non-limiting examples of Trichoderma strains include Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma longibrachiatum, Trichoderma longibrachiatum, Trichoderma longibrachiatum. ), Trichoderma virens, Trichoderma citrinobiride, and Trichoderma virides, as well as alternative tangible / telemorphic forms.

The recombinant microbial host cells provided herein are capable of sporulation or sporulation deficiency.

The recombinant microbial host cells provided herein can be derived from wild-type microbial cells or gene variants thereof (eg, mutants).

The recombinant microbial host cells provided herein can be derived from a Food and Drug Administration Pass Certificate (GRAS) Industrial Strain.

The recombinant microbial host cells provided herein are capable of having a high exogenous secretory protein / biomass ratio. In some such embodiments, the ratio is greater than about 1: 1; greater than about 2: 1; greater than about 3: 1; greater than about 4: 1; about 5: 1. Greater than, greater than about 6: 1, greater than about 7: 1, or greater than about 8: 1.

In some embodiments, or at least two of esterases (eg, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10). The recombinant microbial host cells provided herein comprising an essentially eliminated or modulated activity of a combination of esterases (at least 11, at least 12, or more) are esterases. Or at least 2 (eg, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 Recombinant trichoderma lysee host cells (ie, recombinant host cells derived from the trichoderma lysey strain), which contain an essentially eliminated or modulated activity of a combination of esterases (or more). Esterase or at least 2 (eg, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least Recombinant black mold host cells (ie, recombinant host cells derived from the black mold strain), esterases containing an essentially eliminated or modulated activity of a combination of 12 or more esterases. Or at least 2 (eg, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 Recombinant trichoderma citrinobilide host cells (ie, recombinant host cells derived from the trichoderma citrinobilide strain), which contain an essentially eliminated or modulated activity of a combination of esterases (or more). And or at least 2 of esterases (eg, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11). Recombinant myseri containing an essentially eliminated or modulated activity of a combination of at least 12 or more esterases) It is selected from the group consisting of offtola thermophila host cells (ie, recombinant host cells derived from the Myseri offtola thermophila strain).

Recombinant Components The recombinant microbial host cells provided herein are capable of producing recombinant components. Non-limiting examples of recombinant components include proteins, lipids, carbohydrates, small molecules, food additives, dietary supplements (eg, vitamins), functional foods, and probiotics.

In some embodiments, the recombinant component is a recombinant protein. In some embodiments, the recombinant protein is a recombinant plant protein. As used herein, the term "plant protein" is at least 80% identical (eg, at least 80%) to the sequence of amino acids in a protein naturally found in plants (ie, a protein native to plant cells). Identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, 100% identical) at least 20 amino acids (eg, at least 20, at least 30, A polypeptide comprising a sequence of at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or at least 150, and usually 250 or less amino acids). Non-limiting examples of plant proteins include citrus, ginkgo, conifer, thread cedar, bean, crobe, cedar, pine, angelica, himeuikyo, coriander, cumin, uikyo, parsley, dill, dandelion, wheatwaragiku, marigold, Yomogi, Benibana, Chamomile, Lettuce, Nigayomogi, Kinsenka, Citrus, Sage, Thyme, Chased, Karasina, Olive, Coffee, Togarashi, Nas, Paprika, Cranberry, Kiwi, Vegetable plants (eg carrots, celery), Targetes , Ezoyomogigiku, Taragon, Sunflower, Ichaxou, Basil, Hisop, Lavender, Lemon Barbena, Marjoram, Seiyoyamahakka, Pachori, Boreihakka, Seiyohakka, Mannenrou, Sesame, Sparemint, Ichigesakurasou, Tsubasa, Togarashi, Pimento , Tomato, blueberry, inuhozuki, petunia, asagao, lilac, jasmine, watermelon, goldfish, psyllium, aritasou, buckwheat, amaranth, fudansou, quinoa, spinach, rubarb, jojoba, citrus, chlorella, marla, hazelnut, canola Chingensai, Rutabaga, Frankinsense, Citrus, Elemi, Timer, Pumpkin, Squash, Uridae, Manioc, Dalbergia, Beanaceae plants (eg, alfalfa, lens bean, legume, clover, pea, hassho bean, frijole bola) roja), black green beans, genus Hagi, kanzo, honey bean, mesquito, locust bean, soybean, peanut, tamarind, wisteria, citrus, chick, galvanzo, koroha, green peas, yellow pea, pea pea, blue bean, soramame), geranium, Pomegranate tree, cotton, okra, neem, fig, mulberry, butterfly, eucalyptus, tea tree, near uri, fruit plants (eg apples, apricots, peaches, plums, pears, citrus peaches), strawberries, blackberries, citrus, cherry , Prune, rose, tangerine, citrus (eg, grapefruit, lemon, lime, orange, daidai, mandarin, tangerine), mango, citrus bergamot, boucu, grape, broccoli, bud cabbage, bean family, cauliflower, citrus abrana, rapeseed (cano) -La), cub, cabbage, cucumber, watermelon, honeydumeron, zucchini, kabanoki, walnut, cassaba, baobab, all-spice, almond, pannoki, byakudan, macadamia, taro, gekkakou, aloe vera, garlic, onion, shalot, Vanilla, Yukka, Bechibar, Getto, Omugi, Grain, Harukon, Ginger, Lemongrass, Karasumugi, Palm, Pineapple, Rice, Limegi, Morokoshi, Raikomugi, Ukon, Yamanoimo, Bamboo, Omugi, Kayupte, Kanna, Cardamon, Corn , Wheat, cinnamon, sassafras, linderabenzoin, laurel, avocado, ylang ylang, mace, nutmeg, wasabi noki, tousa, oregano, coriander leaves, charville, ezonegi, aggregated fruits, grain plants, herbaceous plants, leafy vegetables, non-grain beans Plants, nuts, succulents, terrestrial plants, aquatic plants, genus Dalbergia, millet, nuclear fruits, isolated fruits, flowering plants, non-flowering plants, cultured plants, wild plants, trees, shrubs, flowers, grasses, herbaceous plants, Includes brushes, liana, cactus, tropical plants, subtropical plants, temperate plants, and their derivatives and hybrid-derived proteins.

In some embodiments, the plant protein is pea protein (eg, legumin, vicilin, covicilin). In another embodiment, the plant protein is a potato protein (eg, tuberin, protease inhibitor Noteito II).

In some embodiments, the recombinant protein is a recombinant animal protein. As used herein, the term "animal protein" is at least 80% identical (eg, at least 80%) to the sequence of amino acids in a protein naturally found in animals (ie, a protein native to animal cells). Identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, 100% identical) at least 20 amino acids (eg, at least 20, at least 30, A polypeptide comprising a sequence of at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or at least 150, and usually 250 or less amino acids). Non-limiting examples of animal proteins include proteins from insects (eg, flies), mammals (eg, cows, sheep, goats, rabbits, pigs, humans), or birds (eg, chickens). In some embodiments, the animal protein is a structural protein (eg, collagen, tropoelastin, elastin). In another embodiment, the animal protein is egg protein (eg, egg white albumin).

In yet another embodiment, the recombinant protein is a recombinant milk protein. As used herein, the term "milk protein" is at least 80% identical to the sequence of amino acids in proteins naturally found in milk produced in mammals (ie, proteins native to mammalian-produced milk). At least 20 amino acids (eg, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, 100% identical). For example, a polypeptide comprising a sequence of at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or at least 150, and usually 250 or less amino acids. say.

Recombinant milk protein can be recombinant whey protein or recombinant casein. As used herein, the term "whey protein" or "casein" is at least 80% identical (eg, at least 80% identical) to the sequence of amino acids in the natural whey protein or casein, respectively. At least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, 100% identical) at least 20 amino acids (eg, at least 20, at least 30, at least 40, at least A polypeptide comprising a sequence of 50, at least 60, at least 70, at least 80, at least 90, at least 100, or at least 150, and usually 250 or less amino acids). Non-limiting examples of whey proteins include α-lactalbumin, β-lactalbumin, lactoferrin, transferrin, serum albumin, lactoperoxidase, and glycomacropeptides. Non-limiting examples of casein include β-casein, γ-casein, κ-casein, α-S1 casein, and α-S2 casein. Non-limiting examples of nucleic acid sequences encoding milky proteins and caseins are disclosed in Patent Document 1 submitted on August 21, 2015 and Patent Document 2 submitted on August 25, 2017, as described above. The patent document is thereby incorporated herein in its entirety.

Recombinant milk proteins include cattle, humans, baboons, goats, squirrels, camels, horses, donkeys, fox monkeys, pandas, guinea pigs, squirrels, bears, mackerel, gorillas, chimpanzees, white goats, monkeys, apes, cats, dogs, wallabies, Can be derived from any mammalian species including, but not limited to, rats, mice, elephants, opossums, rabbits, whales, baboons, gibbons, orchid utans, mandolin, pigs, wolves, foxes, lions, tigers, and gorillas. Is.

Recombinant milk proteins can lack epitopes that can elicit an immune response in humans or animals. Such recombinant milk proteins are particularly suitable for use in food products.

Recombinant milk proteins can have post-translational modifications. As used herein, the term "post-translational modification" or its acronym "PTM" refers to the covalent bond of a chemical group to a protein after protein biosynthesis. PTM can occur on the amino acid side chain of a protein or at its C or N terminus. Non-limiting examples of PTMs are glycosylation (ie, via C-bonds, N-bonds, or O-bonds, or gripipation (ie, addition of glycosylphosphatidylinositol anchors) or glycosylation phosphate (ie, phosphoserine). Glycosylated groups (ie, monosaccharides, disaccharides, polysaccharides, linear glycans, branched glycans, glycans with galf residues, sulfuric acid and / or glycans with phosphate residues, mediated by the phosphate of the). D-glucose, D-galactose, D-mannose, L-fucose, N-acetyl-D-galactoseamine, N-acetyl-D-glucoseamine, N-acetyl-D-neuraminic acid, galactoflanose, phosphate diester, N-Acetylglucosamine, N-acetylgalactosamine, sialic acid, and combinations thereof; covalently bonded to proteins (see, eg, Non-Patent Document 6)), phosphorylation (ie, covalently bonded phosphate groups to proteins),. Alkylation (ie, the covalent bond of the alcan group (eg, the methane group in methylation) to the protein), and lipidation (ie, the lipid group (eg, prenylation and isoprenylation in the isoprenide group) (eg, farnesylation). Farnesol group in, geraniol group in geranylation, geranyl geraniol in geranyl geranylation, fatty acid group in fatty acid acylation (eg, myristic acid in myristoylation, palmitic acid in palmitoylation), glycosylation in grippyation. Phosphatidyl inositol anchors)), hydroxylation (ie, covalent bonds of hydroxide groups), SUMOization (ie, binding of small ubiquitin-like modifiers (or SUMOs) proteins to proteins), nitrosylation (ie, NO groups). Binds to proteins), as well as tyrosine nitrates (ie, binding of nitrate groups to proteins to tyrosine residues). The PTM of the recombinant milk protein can be a natural PTM, an unnatural PTM, or a mixture of at least one natural PTM and at least one unnatural PTM. As used herein, "intrinsically disordered" refers to a difference in one or more positions of one or more PTMs (eg, glycosylation, phosphorylation) in a protein and / or a naturally occurring protein (ie, ie). , A protein having "natural PTM"), which refers to the difference in the type of one or more PTMs at one or more positions in the protein.

Recombinant milk proteins can have milk protein repeats. As used herein, the term "milk protein repeat" is at least 80% identical to the amino acid sequence found in proteins found in mammalian-produced milk (eg, milky protein, casein) (eg, at least 85%, At least 90%, at least 95% identical, at least 99% identical), not once in recombinant milk protein (eg, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times , At least 15 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 75 times, at least 100 times, at least 150 times, or at least 200 times). Milk protein repeats may contain at least 10, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, or at least 150, and usually no more than 200 amino acids. Milk protein repeats in recombinant milk proteins can be continuous (ie, without intervening amino acid sequences) or discontinuous (ie, with intervening amino acid sequences). When present discontinuously, the intervening amino acid sequence may play a passive role in providing molecular weight without introducing unwanted properties, or certain properties (eg, soluble, biodegradable). , Binding to other molecules) may play an active role.

In some embodiments, the recombinant protein is a recombinant algal protein. As used herein, the term "algal protein" is at least 80% identical (eg, at least 80% identical) to the sequence of amino acids in a protein naturally found in algae (ie, a protein native to algae cells). At least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, 100% identical) at least 20 amino acids (eg, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or at least 150, and usually 250 or less amino acids).

In some embodiments, the recombinant protein is a recombinant fungal protein. As used herein, the term "fungal protein" is at least 80% identical (eg, at least 80% identical) to the sequence of amino acids in a protein naturally found in a fungus (ie, a protein native to fungal cells). At least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, 100% identical) at least 20 amino acids (eg, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or at least 150, and usually 250 or less amino acids).

In some embodiments, the recombinant protein is a recombinant microbial protein. As used herein, the term "microbial protein" is at least 80% identical (eg, at least 80% identical) to the sequence of amino acids in a protein naturally found in a microorganism (ie, a protein native to microbial cells). At least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, 100% identical) at least 20 amino acids (eg, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, or at least 150, and usually 250 or less amino acids).

General Methods Recombinant microbial host cells capable of producing recombinant components can be obtained using methods known in the art. Recombinant microbial host cells typically contain recombinant polynucleotides (eg, recombinant vectors) that encode recombinant components. Recombinant polynucleotides can be prepared by any suitable method known in the art, including but not limited to direct chemical synthesis and cloning.

Recombinant polynucleotides typically contain one or more expression cassettes, each expression cassette being: a promoter (eg, a fungal promoter), a signal sequence as needed (ie, a nascent protein is synthesized). A sequence encoding a peptide that mediates the delivery of a nascent protein bound to a peptide to the outside of a cell), a sequence encoding a recombinant protein, and a termination sequence (or multiple termination sequences). The promoter is operably linked to the signal sequence as needed in a sense orientation (ie, the promoter and the subsequent sequences encoding the signal sequence and protein as needed are the signal sequences and proteins as needed by the promoter. Arranged to be effective in regulating the transcription of the sequence encoding the recombinant protein), the signal sequence as needed is operably linked to the sequence encoding the recombinant protein in a sense orientation (ie). , The signal sequence and the sequence encoding the recombinant protein are arranged so that transcription and translation produce the recombinant protein containing the functional signal sequence), and the termination sequence acts on the sequence encoding the recombinant protein. As ligable linkage (ie, the sequence encoding the recombinant protein and the termination sequence) is effective for the terminator to terminate the transcription of the signal sequence and the sequence encoding the recombinant protein as required. Have been placed).

The promoter may be any suitable promoter that is functional in the recombinant microbial host cell. In some embodiments, the promoter is a constitutive promoter. In other embodiments, the promoter is an inducible or inhibitory promoter (eg, glucose, galactose, lactose, sucrose, cellulose, sophorose, gentiobiose, sorbose, a disaccharide that induces a cellulase promoter, starch, tryptophan, or phosphoric acid. A promoter that is induced or suppressed in the presence of salt). Non-limiting examples of promoters suitable for use in recombinant filamentous fungus host cells include the following proteins: glucoamylase (eg, Aspergillus aspergillus, Aspergillus aspergillus, Aspergillus japonicus, Aspergillus tubingensis, Aspergillus. Fetidas, or Aspergillus carbonarius (glaA of Aspergillus carbonarias), aspergillus (eg, Aspergillus TAKA amylases, Aspergillus neutral alpha amylases, Aspergillus acid stable alpha amylases, fungal α-amylases (amy), bacterial alpha amylases). Proteas (eg, Aspergillus aspergillus protease, Aspergillus alkali protease, Aspergillus oxysporum tripsin-like protease, Trichoderma lyseei protease), Lipase (eg, Aspergillus miehei lipase), Isomelase (eg, Aspergillus) Phosphoric acid isomerase, fungal triose phosphate isomerase (tpi), yeast triose phosphate isomerase), acetoamidase (eg, Aspergillus aspergillus or Aspergillus or other fungal acetamidase (amdS)), dehydrogenase (eg, fungal alcohol dehydrogenase (adhA)) ), Fungal glyceraldehyde-3-phosphate dehydrogenase (gpd), yeast alcohol dehydrogenase), xylanase (eg, fungal xylanase (xlnA), Trichoderma xylanases (xyn1, xyn2, bxl1)), kinase (eg, fungal xylanase (xlnA), trichoderma xylanases) , Yeast 3-phosphoglycerate kinase), hydrolase (eg, fungal cellobiohydrolase I (cbh1), trichodermahydrolase (cbh2, egl1, eggl2)), phosphatase (eg, fusalic acid phosphatase), and other fungal proteins (eg, fusalic acid phosphatase). , Fungus end α-L-arabinase (abnA), Fungus α-L-arabinofuranosidase A (abfA), Fungus α-L-arabinofuranosidase B (abfB), Fungal phytase, Fungal ATP synthesizer, Fungal subunit 9 (oliC), fungal spore-forming specific protein (Spo2), fungal SSO, yeast alcohol oxidase, yeast lactase, Aspergillus aspergillus CPC1 , Pseudo-focal trpC, fungal chitin-degrading enzymes (eg, endo- and exo-chitinase, beta-glucanase), fungal VAZMP-related protein (VAP), fungal translation elongation factor (TEF1), fungal DNA damage response protein (DDRP) , Fungi (eg, Fuzarium or Akapankabi) Hexagonal peroxysomes (Hex1), Fungi (eg, Akapankabi) Catalase) and recombinant filamentous fungi of genes encoding any of the other proteins produced at high levels in host cells. Includes promoters and their functional parts.

Non-limiting examples of promoters suitable for use in recombinant bacteria or yeast host cells include the following proteins: LAC4, T7 polymerase, TAC, GAL1, λPL, λPR, beta lactamase, spa, CYC1, TDH3, GPD, TEF1, ENO2, PGL1, GAP, SUC2, ADH1, ADH2, HXT7, PHO5, CLB1, AOX1, cellulase, amylases, proteases, xylinase, and any other protein produced at high levels in recombinant filamentous fungal host cells. Includes the promoter of the gene encoding either, and its functional portion.

In some embodiments, the promoter is a promoter of a stress (eg, heat shock) response gene (eg, hac1, BIP).

The signal sequence may be any suitable signal sequence that is functional in the recombinant microbial host cell.

Recombinant proteins typically encoded by recombinant polynucleotides are recombinant milk proteins (eg, recombinant milk proteins disclosed herein (eg, recombinant β-lactoglobulin, recombinant α-lactoalbumin, set). Alternate κ casein, recombinant β casein)), recombinant egg protein (eg, ovotransferase (conalbumin), ovomucin, ovalbumin, ovotransferase, lysoteam), recombinant protein required for vitamin production, lipid Any recombinant protein (eg, recombinant components disclosed herein), including flavor-producing small molecules, texture-producing polymers and recombinant proteins required for the production of emulsions, and combinations thereof. Is.

The termination sequence may be any suitable termination sequence that is functional in the recombinant microbial host cell. Non-limiting examples of termination sequences suitable for use in recombinant filamentous fungal host cells are stag beetle (eg, termination sequence of the TAKA amylase gene), black stag beetle (eg, glaA, gpdA, amA, trpC, pdc1, adh1). , AmdS, or the termination sequence of the tef1 gene), Fuzarium oxysporum (eg, the termination sequence of the serine protease (trypsin) gene), Trichoderma lyseei (eg, the termination sequence of the cbh1, pdc1, TEF1, gpd1, xyn1, or adh1 gene). ), Pikia pastris (eg, termination sequence of aox1, gap1, adh1, tef1, tps1, or pgk1 gene), budding yeast (eg, termination sequence of adh1, cyc1, gal1, tef1, pdc1, pgk1, or tps1 gene). Including, but not limited to, any combination of termination sequences, synthetic termination sequences, and sequences listed above. Non-limiting examples of termination sequences suitable for use in recombinant yeast host cells include, but are not limited to, PGK1 and TPS1 termination sequences.

Recombinant polynucleotides can further include additional elements. Non-limiting examples of such additional elements include promoter sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5'and 3'untranslated regions, polyadenylated sequences, introns, origins of replication, operators. (Ie, a sequence of nucleic acids adjacent to the promoter and containing a protein binding domain, to which the repressor protein can bind to reduce or eliminate the activity of the promoter), and selection markers (ie). That is, it contains a protein encoding a protein that can complement the nutrient requirement of filamentous fungal cells, confer antibiotic resistance, or cause color change). Such elements are known in the art. Non-limiting examples of origins of replication include AMA1 and ANSI. Non-limiting examples of suitable selection markers are amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinoslicin acetyltransferase), hph (hyglomycinphosphotransferase), niaD (nitrite reductase). , PyrG / pyrr4 (orotidin 5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranyl acid synthase), and derivatives thereof. In some embodiments, the selectable marker comprises a modification that reduces the production of the selectable marker, thus increasing the number of copies required for the filamentous fungal cells containing the polynucleotide to survive under selection.

In embodiments that include an expression cassette containing two or more recombinant polynucleotides, an operably linked promoter, a signal sequence as required, a sequence encoding a polypeptide termination sequence, and an addition as needed. Elements of can be the same or different between two or more expression cassettes.

Methods for introducing recombinant polynucleotides into microbial cells to obtain recombinant microbial host cells are well known in the art. Non-limiting examples of such methods include calcium phosphate transfection, dendrimer transfection, liposome transfection (eg, cationic liposome transfection), cationic polymer transfection, cell squeezing, sonoporation, light. Includes transfection, protoplast fusion, impalefection, hydrodynamic delivery, gene guns, magnetfection, viral transfection, electrical perforation and chemical transformation (eg, using PEG).

In some embodiments, recombinant polynucleotides transduce, transform, or infect an extrachromosomal expression vector in a recombinant microbial host cell (ie, a nucleic acid native to the host cell and / Or a nucleic acid other than the protein and / or a nucleic acid that expresses the protein in the host cell in a manner that is not unique to the host cell). In other embodiments, the recombinant polynucleotide is stably integrated into the genome (eg, chromosome) of a recombinant microbial host cell. For integration into the genome, recombinant polynucleotides can include sequences for integration into the genome by homologous or illegitimate recombination. In some embodiments, such sequences allow integration at the exact location into the host genome. Recombinant polynucleotides are at least 100, at least 250, at least 500, at least 750 highly homologous to the target sequence in the genome of the recombinant microbial host cell to enhance the potential for homologous recombination. , At least 1000, or at least 10,000 base pairs may be included. Such highly homologous sequences may be non-coding sequences. More than one copy of the recombinant polynucleotide may be inserted into the recombinant microbial host cell to increase production of the recombinant protein.

Recombinant microbial host cells can be any suitable fermentation vessel known in the art (eg, culture plates, shaking flasks, fermenters (eg, stirring tank fermenters, airlift fermenters, bubble tower fermenters, fixed bed bioreactors). , Laboratory fermenters, industrial fermenters, or any combination thereof), as well as any scale (eg, small, large) and process (eg, continuous, batch, fedbatch) known in the art. , Or in the solid state) can also be cultured.

Suitable culture media include any culture medium in which the recombinant microbial host cell can grow and / or remain viable. In some embodiments, the culture medium is carbon, nitrogen (eg, anhydrous ammonia, ammonium sulfate, ammonium nitrate, diammonium phosphate, monoammonium phosphate, ammonium polyphosphate, sodium nitrate, urea, peptone, protein hydrolyzate, An aqueous medium containing a yeast extract) and a phosphate source. The culture medium can further contain inorganic salts, minerals, metals, transition metals, vitamins, emulsifying oils, surfactants, and any other nutrients. Non-limiting examples of suitable carbon sources include monosaccharides, disaccharides, polysaccharides, acetates, ethanol, methanol, glycerol, methane, and combinations thereof. Non-limiting examples of monosaccharides include glucose, fructose, galactose, xylose, arabinose, and combinations thereof. Non-limiting examples of disaccharides include sacrose, lactose, maltose, trehalose, cellobiose, and combinations thereof. Non-limiting examples of polysaccharides include starch, glycogen, cellulose, amylose, hemicellulose, maltodextrin, and combinations thereof. Suitable culture media may be prepared according to compositions available or published from commercial sources (eg, in the United States Culture Cell Lineage Conservation Agency (ATCC) inventory).

Suitable conditions for the production of recombinant components are conditions under which the recombinant microbial host cell can grow and / or remain viable. Non-limiting examples of such conditions are the right pH, the right temperature, the right feed, the right pressure, the right nutrient content (eg, the right carbon content, the right nitrogen content, the right phosphorus). Content), the appropriate dietary supplement content, the appropriate trace metal content, and the appropriate oxidation level.

In some embodiments, the culture medium is a protease capable of preventing the degradation of the recombinant protein (eg, a plant-derived protease), a protease inhibitor that reduces the activity of the protease capable of degrading the recombinant protein, And / or further include sacrificial proteins that can divert protease activity. In some embodiments, the culture medium further comprises an unnatural amino acid for integration with the produced recombinant protein.

Other Genetic Modifications The recombinant microbial host cells provided herein can further comprise a genetic modification that improves the production of recombinant components. Non-limiting examples of such gene modifications are altered promoters, altered kinase activity, altered phosphatase activity, altered protein folding activity, altered protein secretory activity, and altered gene expression induction. Includes route.

The recombinant microbial host cells provided herein can further have reduced or essentially eliminated protease activity to minimize degradation of the recombinant protein (eg, patent literature). 3). Filamentous cells with reduced or essentially eliminated protease activity can be obtained by screening for mutants by methods known in the art or by specific genetic modification.

The recombinant microbial host cells provided herein can further comprise a native or heterologous glycosyltransferase. Non-limiting examples of such endogenous or heterologous glycosyltransferases include fucosyltransferases, galactosyltransferases, glucosyltransferases, xylosyltransferases, acetylases, glucolonyltransferases, glucoronylepimerases, sialyltransferases, sialyltransferases. , Sulfotransferase, β-acetylgalactosaminyltransferase, and N-acetylglucosaminyltransferase.

The recombinant microbial host cells provided herein can further comprise native or heterologous kinases or phosphatases. Non-limiting examples of such natural or heterologous kinases or phosphatases are protein kinase A, protein kinase B, protein kinase C, creatine kinase B, protein kinase C beta, protein kinase G, TmkA, Fam20 kinase (eg, Fam20C). ), ATM, CaM-II, cdc2, cdk5, CK1, CKII, DNAPK, EGFR, GSK3, INSR, p38MAPK, RSK, SRC, phosphotransferase, alkaline phosphatase (eg UniProtKB-O77578), acid phosphatase, and others. (See, for example, Non-Patent Document 7).

Preparations Containing Recombinant Components and Substantially Removed or Modulated Esterase Activity In another embodiment, recombinant components (eg, any of the recombinant components disclosed herein) and corresponding preparations. Esterase activity substantially eliminated or modulated relative to the esterase activity contained in the substance (eg, the activity of any one esterase disclosed herein or at least two esterases disclosed herein. Preparations comprising any combination of activities) are provided herein.

In some embodiments, the preparations provided herein are at least 10%, at least 15%, at least 20%, and at least 25 relative to the esterase activity contained in the recombinant component and the corresponding preparation. %, At least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, It comprises a substantially eliminated or modulated esterase activity that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% reduced. In some such embodiments, the preparations provided herein comprise recombinant components and substantially eliminated esterase activity. In other embodiments, the preparations provided herein are at least 25%, at least 50%, at least 75%, at least 100% of the esterase activity contained in the recombinant component and the corresponding preparation. , At least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1,000% reduced esterase activity. include.

The preparations provided herein may be obtained by purifying the recombinant components produced by the recombinant microbial host cells provided herein from a fermentation broth containing recombinant microbial host cells.

Methods for purifying recombinant components from fermented broth are well known in the art (eg, Protein Purification, JC Janson and L Ryden, Eds., VCH Publicers, New York, 1989; Protein Purification). See Explorer, ELV Harris and S Angel, Eds., IRL Press, Oxford, England, 1989).

Recombinant components are purified based on their molecular weight by, for example, size exclusion / exchange chromatography, ultrafiltration through a membrane, gel permeation chromatography (eg, preparative disc gel electrophoresis), or density centrifugation. May be.

Recombinant components are also purified by isoelectric focusing, anion / cation exchange chromatography, isoelectric focusing (IEF), or reverse phase chromatography, based on their surface charge or hydrophobicity / hydrophilicity. May be good.

The recombinant component may also be purified by, for example, ammonium sulfate precipitation, isoelectric precipitation, detergent, detergent, or solvent extraction based on its solubility.

The recombinant component may also be purified by affinity chromatography, reactive dye, or hydroxyapatite, for example, based on its affinity for another molecule. Affinity chromatography is performed on an antibody that has a specific binding affinity for a recombinant component, or a nickel NTA for a His-labeled recombinant protein, or a lectin for binding to the sugar moiety of a recombinant protein, or a recombinant component. In particular, it may include the use of any other molecule to which it binds. In some embodiments, the recombinant component has a tag that facilitates purification. Non-limiting examples of such tags include epitope tags and protein tags. Non-limiting examples of epitope tags include c-myc, hemagglutinin (HA), polyhistidine (6x-HIS), GLU-GLU, and DYKDDDDK (FLAG) epitope tags. Non-limiting examples of protein tags include glutathione-S-transferase (GST), green fluorescent protein (GFP), and maltose binding protein (MBP). Epitopes or protein tags may be removed after isolation of the recombinant component (eg, via protease cleavage).

In embodiments where the recombinant component is secreted by the recombinant microbial host cell, the recombinant component may be purified directly from the fermentation broth. In other embodiments, the recombinant component may be purified from cytolysis.

The identity of the recombinant components includes high performance liquid chromatography (HPLC), Western blot analysis, Eastern blot analysis, polyacrylamide gel electrophoresis, capillary electrophoresis, enzyme product formation, enzyme substrate disappearance, and 2-dimensional mass spectroscopy. It may be confirmed and / or quantified by method (2D-MS / MS) sequence identification.

In some embodiments, the recombinant component is greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, 60% relative to the other components contained in the fermented broth. Purified to a purity greater than%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 97%, or greater than 99%. In some embodiments, the recombinant component is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, relative to the other components in the preparation than in the fermented broth. It is purified to be at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold rich. In some embodiments, the recombinant component is greater than 30% by weight, greater than 35% by weight, greater than 40% by weight, greater than 45% by weight, greater than 50% by weight, greater than 55% by weight, greater than 60% by weight, greater than 65% by weight. Super, over 70% by weight, over 75% by weight, over 80% by weight, over 85% by weight, over 90% by weight, over 95% by weight, over 97% by weight, or over 99% by weight.

In some embodiments, the preparation is a liquid. In other embodiments, the preparation is solid. In some such embodiments, the preparation is a powder. In some such embodiments, the powder comprises a water content of less than 10% by weight, less than 7% by weight, less than 5% by weight, less than 3% by weight, or less than 1% by weight. The powder may be obtained, for example, by spray drying or concentration via evaporation.

In some embodiments, the preparation comprises a post-treated recombinant component. Post-treatment of the recombinant component involves fragmentation (eg, by chemical means or by exposure to protease enzymes (eg, trypsin, pepsin, carmapepsin)), removal of reactive sites (eg, methionine and / by oxidation). Or removal of reactive sites of tryptophan residues), modulation (eg, via chemical, photochemical, and / or enzymatic strategies), cyclization, biotinylation (ie, binding biotin), and It may include conjugates to other elements (eg, polyethylene glycol, antibodies, liposomes, phospholipids, DNA, RNA, nucleic acids, sugars, disaccharides, polysaccharides, starches, celluloses, detergents, cell walls).

Post-treatment is done in a random or site-specific manner (eg, sulfhydryl groups of cysteine residues (eg, aminoethylation, isothiocyanate formation, maleimide formation, Dha formation, covalent bonds via disulfide bonds, etc.). And for desulfurization); primary amine groups of lysine residues (eg, for binding of activated esters, sulfonyl chlorides, isothiocyanates, unsaturated aldehyde esters, and aldehydes); phenolic of tyrosine residues Hydroxyl groups; removal of specific epitopes (eg, glycan groups) capable of inducing an immune response in humans; in azo electron cyclization may be performed.

Post-treatment includes, but is not limited to, certain chemical and / or physical properties of the recombinant component, including, but not limited to, size, charge, hydrophobicity, hydrophilicity, solvation, protein folding, and chemical reactivity. May change.

Food Products Containing Recombinant In Another Aspect, Contain Recombinant Ingredients Produced by Recombinant Host Cells Provided herein (eg, any of the Recombinant Ingredients Disclosed herein). , Substantially removed or modulated relative to esterase activity in the corresponding food product (eg, activity of any one esterase disclosed herein or disclosed herein). Food products comprising the activity of any combination of at least two esterases) are provided herein. As used herein, the term "food product" refers to domestic animals (eg, dogs, cats), livestock (eg, cows, pigs, horses), and wild animals (eg, non-breeding predatory animals). Refers to products that can be ingested by humans or animals, including. The term includes products that can be combined with or added to one or more other ingredients to produce a food product that can be ingested by humans or animals. In various embodiments, the food products provided herein are required by the US Food and Drug Administration (FDA), the US Department of Agriculture, the European Food Safety Agency, and / or other state or regional food regulators. Meet food safety standards.

In some embodiments, the food products provided herein are at least 0.000075% by weight, at least 0.0001% by weight, 0.00025% by weight, at least 0.0005% by weight, at least 0.00075% by weight. %, At least 0.001% by weight, at least 0.0025% by weight, at least 0.005% by weight, at least 0.0075% by weight, at least 0.01% by weight, at least 0.025% by weight, at least 0.05% by weight , At least 0.075% by weight, at least 0.1% by weight, at least 0.25% by weight, at least 0.5% by weight, at least 0.75% by weight, at least 1% by weight, at least 2.5% by weight, at least 5 %%, at least 7.5% by weight, at least 10% by weight, at least 12.5% by weight, at least 15% by weight, at least 17.5% by weight, at least 20% by weight, at least 22.5% by weight, at least 25% by weight , At least 27.5% by weight, at least 30% by weight, at least 32.5% by weight, at least 35% by weight, at least 37.5% by weight. %%, At least 40% by weight, at least 42.5% by weight, at least 45% by weight, at least 47.5% by weight, at least 50% by weight, at least 52.5% by weight, at least 55% by weight, at least 57.5% by weight , At least 60% by weight, at least 62.5% by weight, at least 65% by weight, at least 67.5% by weight, at least 70% by weight, at least 72.5% by weight, at least 75% by weight, at least 77.5% by weight, at least 80% by weight, at least 82.5% by weight, at least 85% by weight, at least 87.5% by weight, at least 90% by weight, at least 92.5% by weight, at least 95% by weight, at least 97.5% by weight, or 100% by weight %, One or more recombinant components produced by one or more recombinant microbial host cells provided herein (eg, any of the recombinant components disclosed herein). include.

In some embodiments, the food products provided herein are esterase activity (eg, the activity of any one esterase disclosed herein or at least two esterases disclosed herein. Substantially free of any combination of activities).

In some embodiments, the food product is composed primarily or entirely of ingredients from non-animal sources. In an alternative embodiment, the food product consists of components from animal sources, but with components from non-animal sources added. In some such embodiments, the grocery product is 5% to 100% by weight, 90% by weight, 80% by weight, 70% by weight, 60% by weight, 50% by weight, 40% by weight, 30% by weight, Between 20% by weight or 10% by weight; 10% to 100% by weight, 90% by weight, 80% by weight, 70% by weight, 60% by weight, 50% by weight, 40% by weight, 30% by weight, or 20% by weight. Between 20% by weight, 90% by weight, 80% by weight, 70% by weight, 60% by weight, 50% by weight, 40% by weight, or 30% by weight; between 30% by weight and 100% by weight. Between 90% by weight, 80% by weight, 70% by weight, 60% by weight, 50% by weight, or 40% by weight; 40% to 100% by weight, 90% by weight, 80% by weight, 70% by weight, 60% by weight. Between 50% or 50% by weight; between 50% by weight and 100% by weight, 90% by weight, 80% by weight, 70% by weight, or 60% by weight; between 60% by weight and 100% by weight, 90% by weight, 80% by weight. Between 70% or 70% by weight; between 70% by weight and 100% by weight, 90% by weight, or 80% by weight; between 80% by weight and 100% by weight, or between 90% by weight; or between 90% by weight and 100% by weight. Includes ingredients from non-animal sources between%.

In various embodiments, the food product is a food product selected from one of the food product categories defined by the National Health and Nutrition Examination Survey, or is similar to such a food product (ie, food product). It is a substitute).

Non-limiting examples of such food products categories (and non-limiting examples of such food products) include snack foods and gums (eg, snack bars, crackers, salted snacks from grain products, chewing gum). Bread, grain, and pasta (eg, oat bread and rolls, corn bread, corn muffins, tortillas, flour and dry mixes, biscuits, multigrain breads and rolls, whole grain breads and rolls, pasta, rye breads and rolls, coarse Beverages (eg, beer and ale, concentrated beverages, beverages, energy drinks, sports drinks, supplements, soft drinks, carbonated beverages, juices, wines, cocktails, nutritional drinks, nutritional powders); ); Confectionery and desserts (eg cakes, candies, chips, cookies, cobras, pastries, ice cream or ice cream sticks, muffins, pies, sugar substitutes or substitutes, syrups, honey, jelly, jams, preserves, salads, crepes , Denish, breakfast pastry, donuts); Breakfast foods (eg, grains, cereals, rice, French toast, pancakes, waffles, coffee cakes); Eggs (eg, egg dishes, egg soup, mixtures made of egg whites) , Egg substitutes, mixtures made from egg substitutes); dairy products; salad dressings, oils, sauces, seasonings (eg cooking fat, vegetable oils, salad dressings, tomato sauce, gravy); potatoes (Eg potato salad, potato soup, chips and sticks, fried potatoes, mashed potatoes, stuffed potatoes, puffs); and soups (eg vegetable soup, vegetable broth), meals, main dishes, proteins (eg meat substitutes) , And seafood included.

In some such embodiments, the food products provided herein are dairy products or are similar to dairy products (ie, dairy products). ) It is a substitute). The term "dairy product" as used herein is milk (eg, whole milk (at least 3.25% milk fat), partially defatted milk (1% to 2% milk fat). ), Non-fat milk (less than 0.2% milk fat), cooking milk, condensed milk, flavored milk, goat milk, sheep milk, powdered milk, sugar-free condensed milk, milk foam), and, but not limited to, yogurt (eg, milk foam). , Whole milk yogurt (at least 6 grams of fat per 170 g), low fat yogurt (fat between 2 and 5 grams per 170 g), non-fat yogurt (0.5 g or less per 170 g), Greek yogurt (fat less than 0.5 g) Drained yogurt from which milk has been removed, whipped yogurt, goat milk yogurt, rabneh (rabne), sheep milk yogurt, yogurt drink (eg, whole milk kefia, low fat milk kefia), Lassie), cheese (eg milky cheese, eg ricotta; pasta filata cheese, eg mozarella; semi-soft cheese, eg Huberty and Muenster; semi-hard cheese, eg Swiss and Jarsberg; hard cheese, eg cheddar and palmezan; washed card Cheeses such as Colby and Monterey Jack; soft aged cheeses such as bristle and camambert; fresh cheeses such as cottage cheese, feta cheese, cream cheese, and curd; process cheese; process cheese foods; process cheese products; process cheese spreads; enzyme-modulated Rated cheese; cold-packed cheese), daily-based sauces (eg, raw, frozen, refrigerated, or can be stored at room temperature), daily spreads (eg, low-fat spreads, low-fat butter), creams (eg, low-fat spreads, low-fat butter) For example, dry cream, heavy cream, light cream, whipping cream, half and half, powdered milk for coffee, cream for coffee, sour cream, creme friche), frozen confectionery (eg ice cream, smoothies, milk shakes). , Frozen yogurt, sundae, gelato, custard), daily dessert (eg raw, chilled or frozen), butter (eg whipped butter, fermented butter), Dairy powder (eg, whole milk powder, defatted milk powder, fat-filled milk powder (ie, milk powder containing vegetable fat instead of all or some animal fat), infant formulas, milk Protein concentrate (ie, at least 80% by weight protein content), milk protein isolate (ie, at least 90% by weight protein content), milky protein concentrate, milky protein isolate, demineralized milk Clean protein concentrate, demineralized milk clean protein concentrate, β-lactoglobulin concentrate, β-lactoglobulin isolate, α-lactoalbumin concentrate, α-lactoalbumin isolate, glycomacropeptide concentrate, glyco Macropeptide isolates, casein concentrates, casein isolates, nutritional supplements, textureizing blends, flavoring blends, colorant blends), pre-prepared beverages or pre-prepared mixed products (eg, raw, refrigerated, or Refers to dairy products including puddings, gels, chewables, crisps, and bars) that can be stored at room temperature, daily protein beverages, weight loss beverages, nutritional beverages, sports recovery beverages, and energy drinks).

The term "food substitute" (eg, "dairy product substitute"), as used herein, is similar to traditional food products (eg, of traditional food products). Refers to food products (which can be used instead). Such similarities may be due to any physical, chemical, or functional attribute. In some embodiments, the similarity between the food products provided herein and conventional food products is due to physical attributes. Non-limiting examples of physical attributes include color, shape, mechanical properties (eg hardness, G'storing modulus value, shape retention, cohesiveness, texture (ie, perception (eg, mouthfeel, fat, etc.)). Mechanical properties), viscosity, and crystallinity that correlate with creaminess, homogeneity, abundance, smoothness, thickness) are included. In some embodiments, the food products and products provided herein and The similarity of traditional food products is due to their chemical / biological attributes. Non-limiting examples of chemical attributes include nutrient content (eg, amino acid type and / or amount (eg, PDCAAS score). ), Fat type and / or amount, Carbohydrate type and / or amount, Mineral type and / or amount, Vitamin type and / or amount), pH, digestibility, shelf life, hunger and / or satiety In some embodiments, the similarities between the food products provided herein and conventional food products are due to functional attributes. Non-functional attributes. Limited examples are gelling / cohesive behavior (eg, gelling ability (ie, physical and / or chemical conditions (eg, stirring, temperature, pH, ionic intensity, protein concentration, sugar concentration, ionicity)). The time required to form a gel with maximum strength in response to (ie) (ie, a protein network with a space filled with a solvent bound to a protein molecule by hydrogen binding), cohesive capacity (ie, physical and). / Or the ability to form precipitates in response to chemical conditions (ie, strong interactions between protein molecules and strong protein networks based on solvent elimination), gel strength (ie, force / unit area (eg, eg). The strength of the formed gel as measured by Pascal (Pa)), the water retention capacity during gelation, the deliquescent during gelation (ie, the leaching of water over time)), the foaming behavior (eg, leaching of water over time). Foaming capacity (ie, the amount of air retained in response to physical and / or chemical conditions), foam stability (ie, half-life of bubbles formed in response to physical and / or chemical conditions) ), Foam spread), thickening ability, versatility of use (ie, ability to use food products in different modes and / or to obtain other diverse compositions from food products; eg milk Ability to produce derivative products, such as food products that resemble yogurt, cheese, cream, and butter) , And the ability to form protein dimers.

In various embodiments, the food products provided herein are dairy substitutes (ie, milk-like), yogurt substitutes (ie, yogurt-like), cheese substitutes. (Ie, similar to cheese), frozen dessert alternatives (ie, similar to frozen desserts (eg, ice cream)), or any other dairy product alternative (ie). , Which are similar to dairy products (eg, any dairy product disclosed herein).

Milk protein component In some embodiments, the food product (eg, dairy product, egg product) comprises a milk protein component, the milk protein component comprises a recombinant milk protein, and the activity of the esterase is substantially. A milk protein that has been removed or not produced according to the methods provided herein (ie, not produced by a method comprising at least one step in which the activity of the esterase is substantially removed or modulated). Modulated compared to the esterase activity of the component. As used herein, the term "milk protein component" is a mixture of a subset of milky protein or a subset of casein or a subset of milky protein and a subset of casein (ie, milky protein concentrate or casein casein). Refers to a component consisting of concentrates, or sodium casein, or acid casein, or derived from some, if not all, proteins present in milk protein concentrates or milk protein isolates. The term is that milk protein in a milk protein component is the only milk protein contained in a food product (ie, the food product does not contain any other milk protein other than the milk protein that makes up the milk protein component). It further implies that.

In some embodiments, the food products provided herein are 0.1% to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight. %, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight, 35% by weight, 30% by weight, 25% by weight, 20% by weight, 15% by weight, 14% by weight, 13% by weight, 12% by weight, 11% by weight, 10% by weight, 9% by weight, 8% by weight, 7% by weight, 6% by weight, 5% by weight, 4% by weight, 3% by weight, 2% by weight, 1% by weight. %, 0.9% by weight, 0.8% by weight, 0.7% by weight, 0.6% by weight, 0.5% by weight, 0.4% by weight, 0.3% by weight, or 0.2% by weight. Between; 0.2% by weight to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50. Weight%, 45% by weight, 40% by weight, 35% by weight, 30% by weight, 25% by weight, 20% by weight, 15% by weight, 14% by weight, 13% by weight, 12% by weight, 11% by weight, 10% by weight. , 9% by weight, 8% by weight, 7% by weight, 6% by weight, 5% by weight, 4% by weight, 3% by weight, 2% by weight, 1% by weight, 0.9% by weight, 0.8% by weight, 0 Between 0.7% by weight, 0.6% by weight, 0.5% by weight, 0.4% by weight or 0.3% by weight; 0.3% by weight to 100% by weight, 95% by weight, 90% by weight, 85 Weight%, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight, 35% by weight, 30% by weight, 25% by weight. , 20% by weight, 15% by weight, 14% by weight, 13% by weight, 12% by weight, 11% by weight, 10% by weight, 9% by weight, 8% by weight, 7% by weight, 6% by weight, 5% by weight, 4%. Weight%, 3% by weight, 2% by weight, 1% by weight, 0.9% by weight, 0.8% by weight, 0.7% by weight, 0.6% by weight, 0.5% by weight or 0.4% by weight Between: 0.4% by weight to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50. Weight%, 45% by weight, 40% by weight, 35% by weight, 30% by weight, 25% by weight, 20% by weight, 15% by weight, 14% by weight, 13% by weight, 12% by weight, 11% by weight, 10% by weight. , 9% by weight, 8% by weight, 7% by weight, 6% by weight, 5% by weight, 4% by weight, 3% by weight, 2% by weight, 1% by weight, 0.9% by weight, 0.8% by weight, 0 Between 7% by weight, 0.6% by weight or 0.5% by weight; 0.5% to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight, 35% by weight, 30% by weight, 25% by weight, 20% by weight, 15% by weight, 14% by weight, 13% by weight, 12% by weight, 11% by weight, 10% by weight, 9% by weight. %, 8% by weight, 7% by weight, 6% by weight, 5% by weight, 4% by weight, 3% by weight, 2% by weight, 1% by weight, 0.9% by weight, 0.8% by weight, 0.7% by weight. % Or 0.6% by weight; 0.6% to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight. %, 55% by weight, 50% by weight, 45% by weight, 40% by weight, 35% by weight, 30% by weight, 25% by weight, 20% by weight, 15% by weight, 14% by weight, 13% by weight, 12% by weight, 11% by weight, 10% by weight, 9% by weight, 8% by weight, 7% by weight, 6% by weight, 5% by weight, 4% by weight, 3% by weight, 2% by weight, 1% by weight, 0.9% by weight, Between 0.8% by weight or 0.7% by weight; 0.7% by weight to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight %, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight, 35% by weight, 30% by weight, 25% by weight, 20% by weight, 15% by weight, 14% by weight, 13% by weight, 12% by weight, 11% by weight, 10% by weight, 9% by weight, 8% by weight, 7% by weight, 6% by weight, 5% by weight, 4% by weight, 3% by weight, 2% by weight, 1% by weight, 0. Between 9% or 0.8% by weight; 0.8% to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight, 35% by weight, 30% by weight, 25% by weight,
20% by weight, 15% by weight, 14% by weight, 13% by weight, 12% by weight, 11% by weight, 10% by weight, 9% by weight, 8% by weight, 7% by weight, 6% by weight, 5% by weight, 4% by weight. %, 3% by weight, 2% by weight, 1% by weight or 0.9% by weight; 0.9% by weight to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight. %, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight, 35% by weight, 30% by weight, 25% by weight, 20% by weight, 15% by weight, 14% by weight, 13% by weight, 12% by weight, 11% by weight, 10% by weight, 9% by weight, 8% by weight, 7% by weight, 6% by weight, 5% by weight, 4% by weight%, 3% by weight, 2% by weight. % Or 1% by weight; 1% to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight. %, 50% by weight, 45% by weight, 40% by weight, 35% by weight, 30% by weight, 25% by weight, 20% by weight, 15% by weight, 14% by weight, 13% by weight, 12% by weight, 11% by weight, Between 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3% or 2%; 2% to 100%, 95%, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight, 35% by weight, 30% by weight. %, 25% by weight, 20% by weight, 15% by weight, 14% by weight, 13% by weight, 12% by weight, 11% by weight, 10% by weight, 9% by weight, 8% by weight, 7% by weight, 6% by weight, Between 5% by weight, 4% by weight or 3% by weight; 3% to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight, 35% by weight, 30% by weight, 25% by weight, 20% by weight, 15% by weight, 14% by weight, 13% by weight, 12% by weight. %, 11% by weight, 10% by weight, 9% by weight, 8% by weight, 7% by weight, 6% by weight, between 5% by weight or 4% by weight; 4% to 100% by weight, 95% by weight, 90% by weight. %, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight, 35% by weight, 30% by weight, 25% by weight, 20% by weight, 15% by weight, 14% by weight, 13% by weight, 12% by weight, 11% by weight%, 10% by weight, 9% by weight , 8% by weight, 7% by weight, 6% by weight or 5% by weight; 5% to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight. , 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight, 35% by weight, 30% by weight, 25% by weight, 20% by weight, 15% by weight, 14% by weight, 13 Between 6% by weight, 12% by weight, 11% by weight, 10% by weight, 9% by weight, 8% by weight, 7% by weight or 6% by weight; 6% to 100% by weight, 95% by weight, 90% by weight, 85 Weight%, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight, 35% by weight, 30% by weight, 25% by weight. Between 20% by weight, 15% by weight, 14% by weight, 13% by weight, 12% by weight, 11% by weight, 10% by weight, 9% by weight, 8% by weight or 7% by weight; 7% by weight to 100% by weight. , 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight, 35. Between 9% by weight, 30% by weight, 25% by weight, 20% by weight, 15% by weight, 14% by weight, 13% by weight, 12% by weight, 11% by weight, 10% by weight, 9% by weight or 8% by weight; Weight% to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight ,
Between 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12%, 11%, 10% or 9%; 9% to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight. %, 40% by weight, 35% by weight, 30% by weight, 25% by weight, 20% by weight, 15% by weight, 14% by weight, 13% by weight, 12% by weight, between 11% by weight or 10% by weight; 10% by weight. % To 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, Between 40%, 35%, 30%, 25%, 20%, 15%, 14%, 13%, 12% or 11%; 11% to 100%, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight, 35% by weight. %, 30% by weight, 25% by weight, 20% by weight, 15% by weight, 14% by weight, 13% by weight or 12% by weight; 12% to 100% by weight, 95% by weight, 90% by weight, 85% by weight. %, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight, 35% by weight, 30% by weight, 25% by weight, Between 20%, 15%, 14% or 13%; 13% to 100%, 95%, 90%, 85%, 80%, 75%, 70%, Between 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15% or 14%; 14% to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight %, 40% by weight, 35% by weight, 30% by weight, 25% by weight, 20% by weight or 15% by weight; 15% to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight. %, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight, 35% by weight, 30% by weight, 25% by weight or 20. Between 20% by weight; 20% by weight to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50. Between 25% by weight, 45% by weight, 40% by weight, 35% by weight, 30% by weight or 25% by weight; 25% to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight. Between 30% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight, 35% by weight or 30% by weight; 30% by weight to 100% by weight, 95% by weight. Weight%, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight, 45% by weight, 40% by weight or 35% by weight. Between; 35% by weight to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight, 55% by weight, 50% by weight. , 45% by weight or 40% by weight; 40% to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight, 60% by weight. , 55% by weight, 50% by weight or 45% by weight; 45% to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight. , 60% by weight, 55% by weight or 50% by weight; 50% to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight. , 60% by weight or 55% by weight; 55% to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight, 70% by weight, 65% by weight or 60% by weight. Between 60% to 100%, 95%, 90%, 85%, 80%, 75%, 70% or 65%; 65% to 100%, 95% Between 70% by weight, 90% by weight, 85% by weight, 80% by weight, 75% by weight or 70% by weight; 70% to 100% by weight, 95% by weight, 90% by weight, 85% by weight, 80% by weight or 75% by weight. Between 75% by weight; between 75% by weight, 95% by weight, 90% by weight, 85% by weight or 80% by weight; between 80% by weight and 100% by weight, 95% by weight, 90% by weight or 85% by weight. Between; or between 85% by weight and 100% by weight, 95% by weight, 90% by weight; and between 90% by weight and 100% by weight. Or contains milk protein components between 95% by weight, or between 95% and 100% by weight.

The milk protein component contained in the food products provided herein is one or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) milk. It may consist of a protein (eg, one or more milky proteins, one or more caseins, or a mixture of one or more milky proteins and one or more caseins) among the milk proteins. At least one is a recombinant milk protein produced by a recombinant microbial host cell (eg, any of the recombinant milk proteins disclosed herein).

In some embodiments, the milk protein component contained in the food products provided herein is one or more (eg, 2, 3, 4, or more) milk protein components (eg, 2, 3, 4, or more). A mixture of β-lactoglobulin, α-lactoalbumin, β-lactoglobulin and α-lactoalbumin, a mixture of two or more β-lactoglobulin with different post-translational modifications (PTM), two with different PTM Or a mixture of more than α-lactoalbumin, a mixture of two or more β-lactoglobulin and α-lactoalbumin with different PTMs, two or more α-lactoalbumin and β-with different PTMs. A mixture of lactoglobulin, a mixture of two or more β-lactoglobulins with different PTMs, and a mixture of two or more α-lactoalbumin with different PTMs; one or more (eg, 2, 3, 4, or more) casein (eg, κ-casein, β-casein, γ-casein, κ-casein and β-casein mixture, κ-casein and γ-casein mixture, β-casein and A mixture of γ-casein, a mixture of two or more κ-casein with different PTMs, a mixture of two or more β-casein with different PTMs, two or more γ-with different PTMs. A mixture of casein, a mixture of two or more κ-casein and β-casein with different PTMs, a mixture of two or more κ-casein and γ-casein with different PTMs, two with different PTMs Or a mixture of more than β-casein and κ-casein, a mixture of two or more β-casein and γ-casein with different PTMs, two or more γ-casein and κ- with different PTMs. A mixture of casein, a mixture of two or more γ-casein and β-casein with different PTMs, two or more κ-casein with different PTMs and / or two or more with different PTMs. A mixture of two or more γ-casein with β-casein and / or different PTMs); or any combination thereof.

The at least one recombinant milk protein may be a single recombinant milk protein. A single recombinant milk protein is a single recombinant milk protein (eg, recombinant β-lactoglobulin or recombinant α-lactalbumin) or a single recombinant casein (eg, recombinant κ-casein or pair). It may be (alternate β-casein or recombinant γ-casein). Alternatively, at least one recombinant milk protein may be two or more recombinant milk proteins. Two or more recombinant milk proteins are two or more recombinant milky proteins (eg, recombinant β-lactoglobulin and recombinant α-lactoalbumin), two or more recombinant casein. (For example, recombinant κ-casein and recombinant β-casein, recombinant κ-casein and recombinant γ-casein, recombinant κ-casein and recombinant β-casein and recombinant γ-casein), or at least one. A mixture of recombinant milky protein and at least one recombinant casein (eg, one or both of recombinant β-lactoglobulin and recombinant α-lactoalbumin and recombinant κ-casein and recombinant β-casein and pairs. It may be a combination with one or two or all of the substituted γ-casein).

In some embodiments, the at least one recombinant milk protein comprises at least one recombinant milk protein having a PTM. In various such embodiments, the PTM may be a natural PTM, a non-natural PTM, or a mixture thereof. In some embodiments, the type and / or number of PTMs of recombinant milk protein imparts the desired attributes to the food products provided herein.

In some embodiments, the milk protein components contained in the food products provided herein are one or more (eg, 2, 3, 4, or more) recombinant milk protein components (eg, 2, 3, 4, or more). For example, a mixture of recombinant β-lactoglobulin, recombinant α-lactalbumin, recombinant β-lactoglobulin and recombinant α-lactoalbumin, a mixture of two or more recombinant β-lactoglobulins with different PTMs. , A mixture of two or more recombinant α-lactalbumin with different PTMs, a mixture of two or more recombinant β-lactoglobulin and recombinant α-lactalbumin with different PTMs, with different PTMs A mixture of two or more recombinant α-lactalbumin and β-lactoglobulins, a mixture of two or more recombinant β-lactoglobulins with different PTMs, and two or more with different PTMs. (Mixed of recombinant α-lactalbumin); one or more (eg, 2, 3, 4, or more) recombinant caseins (eg, recombinant κ-casein, recombinant β-casein, recombinant γ). -Mixed casein, recombinant κ-casein and recombinant β-casein, mixture of recombinant κ-casein and recombinant γ-casein, mixture of recombinant β-casein and recombinant γ-casein, with different PTM 2 Mixtures of recombinant κ-caseins of one or more, mixtures of two or more recombinant β-caseins with different PTMs, mixtures of two or more recombinant γ-caseins with different PTMs, different A mixture of two or more recombinant κ-caseins and recombinant β-caseins with PTMs, a mixture of two or more recombinant κ-caseins and γ-caseins with different PTMs, 2 with different PTMs Mixtures of recombinant β-casein and recombinant κ-casein of one or more, mixtures of recombinant β-casein and γ-casein of two or more with different PTMs, two or more with different PTMs. A mixture of recombinant γ-casein and recombinant κ-casein, a mixture of two or more recombinant γ-casein and β-casein with different PTMs, two or more recombinant κs with different PTMs. -Caseins and / or recombinant β-caseins with two or more different PTMs and / or pairs of two or more with different PTMs A mixture of γ-casein); or any combination thereof.

In some embodiments, the milk protein component contained in the food products provided herein is one or more (eg, 2, 3, 4, or more) recombinant casein proteins (eg, 2, 3, 4, or more). For example, a mixture of recombinant β-lactoglobulin, recombinant α-lactoalbumin, recombinant β-lactoglobulin and recombinant α-lactoalbumin, a mixture of two or more recombinant β-lactoglobulin with different PTMs. , A mixture of two or more recombinant α-lactoalbumin with different PTMs, a mixture of two or more recombinant β-lactoglobulin and recombinant α-lactoalbumin with different PTMs, with different PTMs. A mixture of two or more recombinant α-lactoalbumin and β-lactoglobulin, a mixture of two or more recombinant β-lactoglobulin with different PTMs, and two or more with different PTMs. Recombinant α-lactoalbumin mixture); one or more (eg, 2, 3, 4, or more) recombinant casein (eg, recombinant κ-casein, recombinant β-casein, recombinant γ-). Casein, a mixture of recombinant κ-casein and recombinant β-casein, a mixture of recombinant κ-casein and recombinant γ-casein, a mixture of recombinant β-casein and recombinant γ-casein, two with different PTMs Or a mixture of recombinant κ-casein above it, a mixture of two or more recombinant β-casein with different PTMs, a mixture of two or more recombinant γ-casein with different PTMs, a different PTM A mixture of 2 or more recombinant κ-casein and recombinant β-casein with different PTMs, a mixture of 2 or more recombinant κ-casein and γ-casein with different PTMs, 2 with different PTMs Or a mixture of recombinant β-casein and recombinant κ-casein above it, a mixture of two or more recombinant β-casein and γ-casein with different PTMs, two or more with different PTMs. A mixture of recombinant γ-casein and recombinant κ-casein, a mixture of two or more recombinant γ-casein and β-casein with different PTMs, two or more recombinant κ-with different PTMs. Recombination of two or more recombinant β-casein with casein and / or different PTMs and / or two or more with different PTMs E γ-casein mixture); or any combination thereof.

The milk protein component may further comprise one or more natural milk proteins. In some embodiments, the milk protein component contained in the food products provided herein is one or more (eg, 2, 3, 4, or more) natural casein proteins (eg, 2, 3, 4 or more). , Natural β-lactoglobulin, natural α-lactoalbumin, mixture of natural β-lactoglobulin and natural α-lactoalbumin, mixture of two or more natural β-lactoglobulin with different PTMs, 2 with different PTMs A mixture of one or more natural α-lactoalbumin, a mixture of two or more natural β-lactoglobulin and natural α-lactoalbumin with different PTMs, two or more natural α-with different PTMs. A mixture of lactoalbumin and native β-lactoglobulin, a mixture of two or more natural β-lactoglobulin with different PTMs, and a mixture of two or more natural α-lactolobulin with different PTMs); 1 A mixture of one or more (eg, 2, 3, 4, or more) natural caseins (eg, natural κ-casein, natural β-casein, natural γ-casein, natural κ-casein and natural β-casein). A mixture of natural κ-casein and natural γ-casein, a mixture of natural β-casein and natural γ-casein, a mixture of two or more natural κ-casein with different PTMs, two or more with different PTMs. Natural β-casein mixture, mixture of 2 or more natural γ-casein with different PTM, mixture of 2 or more natural κ-casein and natural β-casein with different PTM, different PTM A mixture of two or more natural κ-casein and natural γ-casein with two or more, a mixture of two or more natural β-casein and natural κ-casein with different PTMs, two or more with different PTMs. A mixture of natural β-casein and natural γ-casein, a mixture of two or more natural γ-casein and natural κ-casein with different PTMs, two or more natural γ-casein with different PTMs and A mixture of native β-casein, two or more natural κ-casein with different PTMs and / or two or more natural β-casein with different PTMs and / or two or more with different PTMs. Natural γ-casein mixture); or any combination thereof nothing.

In some such embodiments, the milk protein component contained in the food products provided herein is between about 10 to about 1 and about 1 to about 10 (eg, from whey protein and casein). , About 10: 1, about 9: 1, about 8: 1, about 7: 1, about 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1, about 1: 1. , About 1: 2, about 1: 3, about 1: 4, about 1: 5, about 1: 6, about 1: 7, about 1: 8, about 1: 9, or about 1:10) It consists of.

In some such embodiments, the milk protein components contained in the food products provided herein are from about 10 to about 1 to about 1: 1 from recombinant whey protein and natural whey protein. Between 10 (eg, about 10: 1, about 9: 1, about 8: 1, about 7: 1, about 6: 1, about 5: 1, about 4: 1, about 3: 1, about 2: 1) , About 1: 1, about 1: 2, about 1: 3, about 1: 4, about 1: 5, about 1: 6, about 1: 7, about 1: 8, about 1: 9, or about 1 pair. It consists of the weight ratio of 10).

In some such embodiments, the milk protein component contained in the food products provided herein is between about 10 to about 1 to about 1 to about 10 (from recombinant casein and natural casein). For example, about 10 to 1, about 9 to 1, about 8 to 1, about 7 to 1, about 6 to 1, about 5 to 1, about 4 to 1, about 3 to 1, about 2 to 1, about 1 pair. 1, about 1: 2, about 1: 3, about 1: 4, about 1: 5, about 1: 6, about 1: 7, about 1: 8, about 1: 9, or about 1:10) It consists of a ratio.

In some such embodiments, the milk protein component contained in the food products provided herein is from about 10 to about 1 to about 1 to about 10 from recombinant whey protein and recombinant casein. Between (eg, about 10 to 1, about 9 to 1, about 8 to 1, about 7 to 1, about 6 to 1, about 5 to 1, about 4 to 1, about 3 to 1, about 2 to 1, About 1: 1, about 1: 2, about 1: 3, about 1: 4, about 1: 5, about 1: 6, about 1: 7, about 1: 8, about 1: 9, or about 1:10 ) By weight ratio.

In some such embodiments, the milk protein component contained in the food products provided herein is between about 10 to about 1 to about 1 to about 10 from natural whey protein and natural casein. (For example, about 10 to 1, about 9 to 1, about 8 to 1, about 7 to 1, about 6 to 1, about 5 to 1, about 4 to 1, about 3 to 1, about 2 to 1, about 1). One to one, about one to two, about one to three, about one to four, about one to five, about one to six, about one to seven, about one to eight, about one to nine, or about one to ten). It consists of a weight ratio.

In some such embodiments, the milk protein components contained in the food products provided herein are from about 10 to about 1 to about 1 to about 10 from recombinant milk proteins and natural milk proteins. Between (eg, about 10 to 1, about 9 to 1, about 8 to 1, about 7 to 1, about 6 to 1, about 5 to 1, about 4 to 1, about 3 to 1, about 2 to 1, about 1: 1, about 1: 2, about 1: 3, about 1: 4, about 1: 5, about 1: 6, about 1: 7, about 1: 8, about 1: 9, or about 1:10) It consists of the weight ratio of.

In all embodiments, the milk protein component consists of only a subset of whey protein, or a subset of casein, or a mixture of a subset of whey protein and a subset of casein. In some embodiments, a subset of whey protein consists of β-lactoglobulin and / or α-lactalbumin. In some embodiments, the subset of casein consists of κ-casein and / or β-casein and / or γ-casein. In various embodiments, a mixture of a subset of whey protein and a subset of casein is from a combination of β-lactoglobulin and / or α-lactalbumin with κ-casein and / or β-casein and / or γ-casein. Become. In some such embodiments, the mixture consists of β-lactoglobulin and κ-casein. In other such embodiments, the mixture consists of α-lactalbumin and κ-casein. In yet other such embodiments, the mixture consists of β-lactoglobulin and α-lactalbumin and κ-casein.

Non-dairy protein component In some embodiments, the food product (eg, dairy product, egg product) comprises a non-dairy protein component, the non-dairy protein component is a recombinant non-dairy protein, and the present specification. Compared to the esterase activity of non-dairy protein components that are not produced according to the method provided in the book (ie, not produced by a method comprising at least one step in which the activity of the esterase is substantially removed or modulated). Contains substantially removed or modulated activity of esterase.

The non-milk protein component contained in the food products provided herein may include non-milk proteins from any source, as well as mixtures of non-milk proteins from various sources. Non-limiting examples of such sources include animals, plants, algae, fungi, and microorganisms.

Non-limiting examples of animals include insects (eg, flies), mammals (eg, cows, sheep, goats, rabbits, pigs, humans), and birds (eg, chickens).

Non-limiting examples of plants include citrus, ginkgo, conifer, thread cedar, biaksin, crobe, cedar, pine, angelica, himeuikyo, coriander, cumin, uikyo, parsley, dill, dandelion, wheatwaragiku, marigold, yomogi. , Benibana, chamomile, lettuce, nigga, citrus, citrus, sage, thyme, chased, caracina, olive, coffee, pepper, eggplant, paprika, cranberry, kiwi, vegetable plants (eg carrots, celery), tagetes, Ezoyomogigiku, Taragon, Sunflower, Ichaxou, Basil, Hisop, Lavender, Lemon Barbena, Marjoram, Seiyoyamahakka, Pachori, Boreihakka, Seiyouhakka, Mandarin, Sesame, Sparemint, Ichigesakurasou, Tsubasa, Togarashi, Pimento Tomato, blueberry, mandarin orange, petunia, asagao, lilac, jasmine, watermelon, goldfish, psyllium, aritasou, buckwheat, amaranth, fudansou, quinoa, spinach, rubarb, jojoba, citrus, chlorella, marla, hazelnut, canola , Rutabaga, Frankinsense, Citrus, Elemi, Timer, Pumpkin, Squash, Uridae, Manioku, Dalbergia, Beanaceae plants (eg, Alfalfa, Lensmame, Mame, Clover, Endomame, Hasshomame, Furihole Boraroya, Black Ingenmame, Citrus, mandarin orange, mandarin orange, mesquito, locust bean, soybean, peanut, tamarind, wisteria, citrus, chick, mandarin orange, coloha, green peas, yellow pea, mandarin orange, blue bean, soramame), geranium, flax, pomegranate, cotton, Okura, neem, fig, citrus, chow, eucalyptus, tea tree, near uri, fruit plant (eg apple, apricot, peach, plum, mandarin orange, mandarin orange), strawberry, blackberry, kistrawberry, cherry, prune, rose, tangerine , Citrus (eg grapefruit, lemon, lime, orange, daidai, mandarin), mango, citrus bergamot, boucu, grapes, broccoli, bud cabbage, amanazuna, cauliflower, sycamore, rapeseed (canola), cub, cabbage, cucumber, Watermelon, mandarin orange, zucchini , Kabanoki, walnut, cassaba, baobab, all spice, almond, pannoki, byakudan, macadamia, taro, geckakou, aloe vera, garlic, onion, charlotte, vanilla, yukka Grass, crow wheat, palm, pineapple, rice, lime tree, morokoshi, rye wheat, corn, yamanoimo, bamboo, oyster, kayupte, canna, cardamon, corn, crow wheat, wheat, cinnamon, sassafras, linderabenzoin, laurel, avocado, Iran , Mace, Natsumeg, Wasabinoki, Tousa, Oregano, Coriander leaf, Charville, Ezonegi, Collective fruit, Grain plant, Herbaceous plant, Leafy vegetable, Non-grain bean plant, Nut plant, Saccharomyces, Terrestrial plant, Aqueous plant, Dalbergia, Millets, nuclear fruits, isolated fruits, flowering plants, non-flowering plants, cultured plants, wild plants, trees, shrubs, flowers, grasses, herbaceous plants, brushes, liana, cactus, tropical plants, subtropical plants, temperate plants, and them. Includes derivatives and hybrids of.

Non-limiting examples of algae include green algae (eg, Chlorella bulgaris, Chlorella pyrennoidossa), brown algae (eg, Alaria marginata, Arinapus). japonicus, Ascophyllum nodosum, Echlonia sp, Eisenia bicyclis, Hijikia fusihorme (Hizikia tailgari algari) ), Laminaria Rongirururisu (Laminaria longirruris), Laminaria Rongishima (Laminaria Longissima), Laminaria Okotenshisu (Laminaria ochotensis), Laminaria Klaus Tonia (Laminaria claustonia), Laminaria Sakkarina (Laminaria saccharina), Laminaria Jigitata (Laminaria digitata) , Laminaria japonica, Macrocystis pyrifera, Petalonia fascia, Cytosifon lome, cystosiphon lome, red algae, etc. Chondrus Oseratsusu (Chondrus ocellatus), Yucheuma-Kottonii (Eucheuma cottonii), Yucheuma-Supinosamu (Eucheuma spinosum), Furuseraria-Fasuchigiata (Furcellaria fastigiata), Gurashiraria - Bursa - pastoris (Gracilaria bursa-pastoris), Gurashiraria-Richenoizu (Gracilaria lichenoides ), Gloiopeltis furcata, Gigartina accullaris, Gigartina bursa-pastoris, Gigartina porphyra, Gigartina pistillata, Gigartina ladura) (Gigartina stellata), Palmaria palmata, Porphyra columbina, Porphyra crispata, Porphyra crispata, Porphyra deuta (Porphyra deuta) (Porphyra porphyra) subobiculata, Porphyra tenera, Porphyridium cruentum, Porphyridium purpureum, Porphyridium purureum, Porphyridium aerugirum (Rhodolla reticleta), Rodella violasea, Rodyla porphyra (Rhodymenia pormata), and derivatives and hybrids thereof.

Non-limiting examples of fungi include Aspergillus nidulans, Aspergillus niger, Aspergillus niger variant Awamori (Aspergillus niger var. Candida albicans, Candida etchellsii, Candida gilliermondii, Candida humilis, Candida humilis, Candida lypica , Candida utilis, Candida versatilis, Chrysosporium lucknowense, Devariomyces Hanzeniis ashbyii, Fusarium gramineum, Fusarium moniliform, Fusarium venenatum, Hansenula polymorpha Seth Marxianus (Kluyveromyces marxianus), Kluyveromyces marxianus variant Lactis, Kluyveromyces marxianus var. raffinosetilizer ), Mucor Miehei, Mucor Miehei variant Kunieet Emerson (Mucor Miehei var.). Cooney et Emerson, Mucor pichilus Lindt, Myceriophthora thermofile, Neurospora crassa, Peni patens), Pichia pastoris, Pichia finlandica, Pichia trehalofila, Pichia koclamae, Pichia koclamae (Pichia minuta), Ogataea minuta, Pichia lindneri, Pichia opuntiae, Pichia thermotrelance (Pichia saria Pichia Pichia guercuum), Pichia Pijuperi (Pichia pijperi), Pichia Suchipuchisu (Pichia stiptis), Pichia methanolica (Pichia methanolica), Rhizopus Nibeusu (Rhizopus niveus), Saccharomyces bayanus (Saccharomyces bayanus), Saccharomyces Betikasu (Saccharomyces beticus ), budding yeast (Saccharomyces cerevisiae), Saccharomyces Chebarieri (Saccharomyces chevalieri), Saccharomyces Deer statics (Saccharomyces diastaticus), Saccharomyces ellipsometry Lee Deus (Saccharomyces ellipsoideus), Saccharomyces Igujigusu (Saccharomyces exiguus), Saccharomyces Furorenchinu Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces pastorianus, Saccharomyces pombe, Saccharomyces saccharomyces Spolidiobolus yeastonii, Spolidiobolus salmonicolol, Sporobiolomyces roseus, Trichoderma lysees saccharomyces roseus, Trichoderma lysees Includes Yarrowia lipolytica), Zygosaccharomyces rouxii, and derivatives and hybrids thereof.

Non-limiting examples of microorganisms include the phylum Firmicutes, the phylum Cyanobacteria (blue-green algae), oscillatriophcideae, Bacillules, lactobacillus. , Euremo (oscillatriales), Bacillus (bacillaceae), Lactobacillus (lactobacillaceae), Acetbacter suboxydans, Acetobacter xylinum (Acetobacter xyline) Platensis, Artrosspira maxima, Bacillus cereus, Bacillus coagulans, Bacillus coagulans, Bacillus bacillus Bacillus licheniformis, Bacillus stearothermophilus, Bacillus subtilis, Escherichia coli, lactobacillus accilus Lacticoccus lactis, Lactococcus lactis Lancefield Group N, Lactobacillus reuteri, Lactobacillus reuteri, Leuconostock citroc dextranicum), Leuconost oc mesenteroides), Micrococcus Rizzo Day Kuti dregs (Micrococcus lysodeikticus), Spirulina, Streptococcus cremoris (Streptococcus cremoris), Streptococcus lactis (Streptococcus lactis), Streptococcus lactis subspecies Jiasechirakuchisu (Streptococcus lactis subspecies diacetylactis), Streptococcus thermophilus (Streptomyces thermophilus), Streptomyces chattanoogensis, Streptomyces griseus, Streptomyces natalensis (Streptomyces natlensis) Seth Oribo black Moge Ness (Streptomyces olivochromogenes), Streptomyces Rubiginosasu (Streptomyces rubiginosus), Tetrahymena thermophila (Tetrahymena thermophile), Tetrahymena Hegewisuki (Tetrahymena hegewischi), Tetrahymena Hiperangurarisu (Tetrahymena hyperangularis), Tetrahymena Marasenshisu (Tetrahymena Malaccensis, Streptomyces, Streptomyces, Streptomyces, Streptomyces, Streptomyces, Streptomyces, Streptomyces, Streptomyces, Streptomyces, Streptomyces, Streptomyces, Streptomyces, Streptomyces, Streptomyces, Streptomyces, Streptomyces, Streptomyces, Streptomyces, Streptomyces, Streptomyces, Streptomyces, Streptomyces, Streptomyces, Streptomyces, Streptomyces.

In some embodiments, the non-milk protein is an animal protein. Non-limiting examples of such proteins include collagen.

In some embodiments, the non-milk protein is a plant protein. Non-limiting examples of such non-dairy protein include pea protein and potato protein.

In some embodiments, the non-milk protein is a fungal protein. Non-limiting examples of such proteins include glucoamylase, xylasese, amylase, glucanase, members of the SUN family (Sim1p, Uth1p, Nca3p, Sun4p), elongation factor 1-alpha, mitochondrial leucyl-tRNA cellulase, alpha. -Amylase, alpha-galactosidase, cellulase, endo-1,4-beta-xylasese, endoglucanase, exo-1,4-beta-xylosidase, glucoamylase, peptidase, aspergylopepsin-1,1,4-beta-D -Glucan cellulase A, alpha-galactosidase A, alpha-galactosidase B, alpha-galactosidase D, alpha-glucuronidase A, beta-galactosidase C, glucan 1,3-beta-glucosidase A, hydrohobin, and glucanend-1, 3-Beta-Glucanase eglC is included.

In some embodiments, the non-milk protein is secreted by a host cell (eg, either a natural or recombinant host cell of an animal, plant, algae, fungus, or microorganism disclosed herein). Derived from non-milk protein. Suitable secreted non-milk proteins are obtained, for example, by obtaining a secretome (ie, for example, culturing animal, plant, algae, fungal, or microbial cells in a liquid culture and removing cells from the cell culture (eg, eg). Secreted proteins obtained by removal (by centrifugation) and concentrating the remaining culture medium as needed; or genomic sequenced and secreted proteins (eg, Mattanovich et al. 2009. Microbial Cell). (See Factories 8:29), whole-cell extract, or fractionated whole-cell extract identified in silico; if necessary, the protein is partially digested, glycosylated, phosphorylated, or Otherwise, they are treated enzymatically; then they are identified (eg, by screening in an assay (eg, high throughput assay) for proteins with similar, identical or different properties compared to, for example, milk proteins). You may. Suitable secreted non-milk proteins may also be identified by screening calcium-rich fractions of proteins with similar or different properties as compared to soy protein, such as milk protein. A non-limiting example of a suitable secreted non-milk protein is provided in PCT / US2017 / 48730, a PCT application filed August 25, 2017. In some embodiments, the non-milk protein is a secreted fungal protein (ie, a protein secreted by a fungus (eg, any of the fungi disclosed herein)).

In some embodiments, the non-milk protein component comprises recombinant non-milk protein. In some such embodiments, the recombinant non-milk protein has an unnatural PTM and / or has no epitope capable of eliciting an immune response in humans or animals.

Other Ingredients The food products provided herein may further comprise one or more other ingredients. For example, in some embodiments, the food products provided herein further comprise lipids. Non-limiting examples of lipids include lipids selected from the group consisting of fats, oils, monoglycerides, diglycerides, triglycerides, phospholipids, and free fatty acids.

Non-limiting examples of oils include vegetable oils (eg sunflower oil, coconut oil, mustard oil, peanut oil, canola oil, corn oil, cottonseed oil, flaxseed oil, olive oil, palm oil, rapeseed oil, benivana oil, sesame oil, large Bean oil, almond oil, beech nut oil, Brazilian nut oil, cashew oil, hazelnut oil, macadamia nut oil, mongongo nut oil, pecan oil, pine nut oil, pistachio nut oil, walnut oil, avocado oil, grape oil), microorganisms Derived oils, algae derived oils, fungal derived oils, marine animal oils (eg, Atlantic fish oil, Pacific fish oil, Mediterranean fish oil, light pressed fish oil), alkaline treated fish oil, heat treated fish oil, light and light brown fish oil (light). And havey brown fish oil), bonito oil, sardine oil, tuna oil, sea bass oil, hariba oil, furai sardine oil, barracuda oil, cod oil, menhaden oil, sardine oil, anchovy oil, capellin oil, Atlantic cod oil, Atlantic herring Oil, Atlantic mackerel oil, Atlantic Menharden oil, salmon oil, and shark oil, squid oil, squid fish oil, octopus oil, oyster oil, lizard oil, whale oil), non-essential oil, essential oil, natural oil, non-hydrogenated oil, There are partially hydride oils, hydride oils (eg, hydride coconut oils), crude oils, semi-refined (also referred to as alkaline refined) oils, ester exchange oils, and refined oils. In some embodiments, long chain oils (eg sunflower oil, corn oil, olive oil, soybean oil, peanut oil, walnut oil, almond oil, sesame oil, cottonseed oil, canola oil, benibana oil, flaxseed oil, palm oil, palm). Nuclear oil, palm fruit oil, coconut oil, babas oil, shea butter, mango butter, cocoa butter, wheat germ oil, rice bran oil, genetically engineered sunflower oil overexpressing 400% oleic acid) were ester-replaced. Combined with short chain triglycerides to produce fatty acid esters. Various combinations of triglycerides and long chain oils can be incorporated to produce a number of different flavor profiles.

Non-limiting examples of monoglycerides and diglycerides include plant-derived monoglycerides and diglycerides (eg, sunflowers, coconuts, peanuts, cotton nuts, olives, palms, rapeseed, benibana, sesame seeds, soybeans, almonds, beech nuts, brazil nuts, etc. There are monoglycerides and diglycerides from cashews, hazelnuts, macadamia nuts, mongongo nuts, pecans, pine nuts, pistachios, walnuts, and avocados. Monoglycerides and diglycerides may contain acyl chains of any of the free fatty acids listed herein. Additional examples of monoglycerides and diglycerides are known in the art.

Non-limiting examples of free fatty acids include butyric acid, caproic acid, capric acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoseric acid, cellotic acid, myristoleic acid, Palmitoreic acid, sapienoic acid, oleic acid, erachiic acid, bacsenic acid, linoleic acid, linoelydic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid, omega-fatty acids (eg, arachidonic acid, omega) -3-Fatty Acids, Omega-6-Fatty Acids, Omega-7-Fatty Acids, Omega-9-Fatty Acids), Fatty Acids with Even Carbons of 4-16 Carbon Length, Monosaturated Acids (especially with 18 carbons), Low interfacial tension (eg, less than 20, less than 15, less than 11, less than 9, less than 7, less than 5, less than 3, less than 2, less than 1, or less than 0.5 dyne / cm, 0.1 to 20, 1 From 15, 2 to 9, 3 to 9, 4 to 9, 5 to 9, 2 to 7, 0.1 to 5, 0.3 to 2, or 0.5 to 1 fatty acid / cm, 0.1, 0 .5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 , 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13 .0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0 , 19.5, or 20.0) fatty acids, butyric acid (4: 0) or caproic acid (6: 0) esterified at sn-3, esterified at sn-1 and sn-2 positions. Medium chain fatty acids (8: 0-14: 0) and 16: 0, fatty acids with stearic acid (18: 0) at the sn-1 position, oleic acid (18: 1) with sn-1 and sn-3. Fatty acids at positions, fatty acids with various carbon atoms (eg, 8-40, 10-38, 12-36, 14-34, 16-32, 18-30, or 20-28 carbon atoms), at least One unsaturated bond (ie, carbon-carbon double or triple bond; eg, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 carbon-carbon double bonds and / Fatty acids containing (or triple bonds), at least one of conjugated unsaturated bonds (ie, carbon-carbon double and / or triple bonds) Fatty acids containing a pair of fatty acids bonded to each other without a methylene (CH2) group between them (eg, 4CH: CHiCH: CHi), as well as derivatives of the above fatty acids (eg, esters (eg, methyl and ethyl esters)). ), Salts (eg, sodium and potassium salts), triglyceride derivatives, diglyceride derivatives, monoglyceride derivatives). Free fatty acids may be saturated or unsaturated. In some embodiments, the free fatty acids are not derived from or produced by mammals. Additional examples of free fatty acids are known in the art.

Non-limiting examples of phospholipids include lecithin phospholipids (eg, soybean lecithin phospholipids, sunflower lecithin phospholipids, cotton lecithin phospholipids, rapeseed lecithin phospholipids, rice bran lecithin phospholipids, corn lecithin phospholipids, flour lecithin phospholipids. Lipids), cardiolipin, ceramide phospholipid, ceramide phosphoethanolamine, glycerophospholipid, phosphatidylic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphosphingolipid, and phosphatidylserine. In some embodiments, phospholipids are not derived from or produced by mammals.

Non-limiting examples of triglycerides include tributyrin, short-chain triglycerides, short-chain triglycerides containing three oleic acids; short-chain triglycerides containing caproic acid; short-chain triglycerides containing caproic acid and butyric acid; There are short chain triglycerides comprising; and one butyric acid, one caproic acid, and one short chain triglyceride containing octanoic acid.

In some embodiments, the food products provided herein are less than 0.3% by weight, less than 0.25% by weight, less than 0.2% by weight, less than 0.15% by weight, 0.1% by weight. Less than or less than 0.05% by weight; 0.05% by weight to 3% by weight, 2.6% by weight, 2.2% by weight, 1.8% by weight, 1.4% by weight, 1.0% by weight Between 0.6% by weight, 0.2% by weight, or 0.1% by weight; 0.1% by weight to 3% by weight, 2.6% by weight, 2.2% by weight, 1.8% by weight, Between 1.4% by weight, 1.0% by weight, 0.6% by weight, or 0.2% by weight; 0.2% by weight to 3% by weight, 2.6% by weight, 2.2% by weight, 1 Between 0.8% by weight, 1.4% by weight, 1.0% by weight, or 0.6% by weight; 0.6% by weight to 3% by weight, 2.6% by weight, 2.2% by weight, 1. Between 8%, 1.4%, or 1.0%; 1.0% to 3%, 2.6%, 2.2%, 1.8%, or 1. Between 4% by weight; between 1.4% by weight and 3% by weight, 2.6% by weight, 2.2% by weight, or 1.8% by weight; between 1.8% by weight and 2.6% by weight, 2.6% by weight Between 2.2% by weight or 2.2% by weight; between 2.2% by weight and 3% by weight, or between 2.6% by weight; or between 2.6% by weight and 3% by weight with free lipid content. ..

In some embodiments, the food products provided herein may be within the desired range as determined by the expert human sensory panel (eg, not disgusting by the expert human sensory panel). Has a free lipid content (with a flavor profile determined by an expert human sensory panel) to have a determined aroma and / or taste.

In some embodiments, the food products provided herein further comprise carbohydrates. Non-limiting examples of carbohydrates are starch, flour, and edible fiber. Non-limiting examples of starch include maltodextrin, inulin, fructooligosaccharides, pectin, carboxymethyl cellulose, guar gum, corn starch, oat starch, potato starch, rice starch, pea starch, and wheat starch. Non-limiting examples of wheat flour include amaranth flour, oat flour, quinoa flour, rice flour, rye flour, sorghum flour, soy flour, wheat flour, and corn flour. Non-limiting examples of edible fiber include bamboo fiber, barley bran, carrot fiber, citrus fiber, corn bran, soluble dietary fiber, insoluble dietary fiber, oat wheat bran, pea fiber, rice bran, head hashs. ), Soy fiber, soy polysaccharide, wheat bran, and wood pulp cellulose. In some embodiments, at least some of the carbohydrates are of plant origin. In some such embodiments, at least some of the carbohydrates are derived from peas.

In some embodiments, the food products provided herein further include natural or artificial sweeteners. Non-limiting examples of sweeteners include stevia, aspartame, cyclamate, saccharin, sucralose, mogloside, brazzein, curculin, erythritol, glycyrrhizin, inulin, isomalt, lacititol, mabinlin, maricitol, mannitol, miraculin, monatin, monerin, osladine. , Pentazine, Sorbitol, Taumatin, Xylitol, Acesulfarm Potassium, Advantage, Ariteme, Aspartame-Acesulfarm, Sodium Cyclamate, Zultin, Glucin, Neohesperidine Dihydrocalcon, Neotame, P-4000, and Carbohydrate-Free Sweetness Contains the agent.

In some embodiments, the food products provided herein further comprise minerals. Non-limiting examples of minerals include fat-soluble minerals, water-soluble minerals, calcium, phosphorus, potassium, sodium, citrate, chloride, phosphate, magnesium, potassium, zinc, iron, molybdenum, manganese, and copper. ..

In some embodiments, the food products provided herein further comprise an emulsifier. Non-limiting examples of emulsifiers include anionic emulsifiers, nonionic emulsifiers, cationic emulsifiers, amphoteric emulsifiers, bioemulsifiers, steric emulsifiers, pickering emulsifiers, glycolipids (eg, trehalose lipids, sophorolipids, ramnolipids, mannosyl). Ellisriol lipid), oligopeptides (eg, gramicidine S, polymyxin), lipopeptides (eg, surfactin), phospholipids, fatty acids, neutral lipids, high molecular weight biosurfactants, amphoteric polysaccharides, lipopolysaccharides, proteins (For example, pea protein, soybean protein, chick bean protein, algae protein, yeast protein, potato protein, lentile protein), mannoprotein, sodium phosphate, stearoyl lactic acid calcium, monoglyceride mono- and diacetyl tartrate, phosphorus Lipids, sorbitan monostearate, magnesium stearate, sodium / potassium / calcium salt of fatty acid, calcium stearoyl lactate, poly-glycerol ester, sorbitan fatty acid ester, acetic acid ester of monoglyceride, lactic acid ester of monoglyceride, citric acid ester of monoglyceride, fatty acid Polyglycerol ester, polyglycerol polylithinolate, propane-1,2-diol ester of fatty acid, sugar ester, sucrose ester of fatty acid, monoglyceride, acetylated monoglyceride, lactic acid monoglyceride, diglyceride, phosphate monoglyceride, diacetyl tartrate, stearic acid. -2-Sodium lactate / calcium, phosphatidammonium, polysorbate, polysorbate-80, carboxymethyl cellulose (CMC), modulated cellulose, citric acid ester, locust bean gum, guar gum, liposan, emalsan, lecithin, surfactant (For example, sorbitan trioleate (Span85), sorbitan tristearate (Span65), sorbitan sesquioleate (Arlacel83), glyceryl monostearate, sorbitan monooleate (Span80), sorbitan monostearate (Span60), monopalmitin. Acid sorbitan (Span40), monolaurate sorbitan (Span20), polyoxyethylene sorbitan tristearate (Tween65), polyoxyethylene sorbitan trioleate (Tween85), polyethylene glyco 400 monostearate, polysorbate 60 (Tween 60), polyoxyethylene monostearate, polysorbate 80 (Tween 80), polysorbate 40 (Tween 40), polysorbate 20 (Tween 20), PEG20 tristearate, PEG20 trioleate, PEG20 monostearate. , PEG20 monooleate, PEG20 monopalmitate, and PEG20 monolaurate sorbitan), and derivatives and mixtures thereof.

In some embodiments, the food products provided herein further comprise antioxidants. Non-limiting examples of antioxidants include natural antioxidants (eg, α-tocopherols (eg, tocopherols contained in bovine milk); low molecular weight thiols (eg, low molecular weight thiols contained in bovine milk). Retinol (eg, retinol contained in bovine milk); TBHQ; carotenoid (eg, carotenoid contained in bovine milk); Vitamin E; Azadiraccha indica extract; Riboflavin; Rosemary extract; Rosemary Phenolic diterpenes contained in the extract (eg, carnosol, carnosic acid); sage extract; ascorbic acid and its salts; lactic acid and its salts; grape residue silage; phenolic compounds contained in the grape residue silage (eg, ferulic acid). ); Soybean (glycine max) extract; Soybean extract contained in isoflavone or polyphenol compound; Garlic (Allium sativum) extract; Sulfur, phenol, flavonoid, or terpenoid compound contained in garlic extract; Phenol (Phenolum vulgare Mill.) Extract; Chamomile (Matricaria recutita L.) extract; Brown algae (eg, Ascophyllum nodosam (Ascophyllum nodosum) fucos, fucos), fucos. Pink pepper essential oil (GEO); mature pink pepper essential oil (MEO); green tea extract; butylated hydroxyanisole (E320); butylated hydroxytoluene (E321); and catechins (eg, catechins contained in green tea extract). Includes acid epigallocatekin, phenolic acid epicatechin, epigallocatekin, C catechin, epicatechin, catechin). In some embodiments, the food products provided herein are less than 2,000 wt ppm. Contains antioxidants.

Food Product Characteristics In some embodiments, the pH of the food product provided herein is 2 to 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6. Between 5.5, 5, 4.5, 4, 3.5, 3, or 2.5; 2.5 to 10, 9.5, 9, 8.5, 8, 7.5, 7, Between 6.5, 6, 5.5, 5, 4.5, 4, 3.5, or 3; 3 to 10, 9.5, 9, 8.5, 8, 7.5, 7, 6 Between 5.5, 6, 5.5, 5, 4.5, 4, or 3.5; 3.5 to 10, 9.5, 9, 8.5, 8, 7.5, 7, 6. Between 5, 6, 5.5, 5, 4.5, or 4; 4 to 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5 Between 5, or 4.5; between 4.5 and 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, or 5; 5 Between 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, or 5.5; 5.5 to 10, 9.5, 9, 8.5, Between 8, 7.5, 7, 6.5, or 6; between 6 and 10, 9.5, 9, 8.5, 8, 7.5, 7, or 6.5; from 6.5 Between 10, 9.5, 9, 8.5, 8, 7.5, or 7; between 7 and 10, 9.5, 9, 8.5, 8, or 7.5; from 7.5 Between 10, 9.5, 9, 8.5, or 8; between 8 and 10, 9.5, 9, or 8.5; between 8.5 and 10, 9.5, or 9; 9 Between 10 and 9.5; or between 9.5 and 10, pH is measured in a solution of deionized water containing a protein content of 4 wt%.

In some embodiments, the food products provided herein are 0.04 to 0.999, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, Between 0.3, 0.2, or 0.1; 0.1 to 0.999, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3 , Or 0.2; between 0.2 and 0.999, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, or 0.3; 0.3 Between 0.999, 0.9, 0.8, 0.7, 0.6, 0.5, or 0.4; 0.4 to 0.999, 0.9, 0.8, 0. Between 7, 0.6, or 0.5; between 0.5 and 0.999, 0.9, 0.8, 0.7, or 0.6; between 0.6 and 0.999, 0. Between 9, 0.8, or 0.7; between 0.7 and 0.999, 0.9, or 0.8; between 0.8 and 0.999, or 0.9; or 0. It has a water activity between 9 and 0.999.

In some embodiments, the food product provided herein is a homogenized food product (ie, a homogenized food product) from 0.11 μm to 15 μm, 12 μm, 10 μm, 8 μm, 6 μm. Between 4, μm, 2 μm, 1 μm, or 0.5 μm; between 0.5 μm and 15 μm, 12 μm, 10 μm, 8 μm, 6 μm, 4 μm, 2 μm, or 1 μm; between 1 μm and 15 μm, 12 μm, 10 μm, 8 μm, 6 μm, 4 μm. Between 2 μm and 15 μm, 12 μm, 10 μm, 8 μm, 6 μm, or 4 μm; between 4 μm and 15 μm, 12 μm, 10 μm, 8 μm, or 6 μm; between 6 μm and 15 μm, 12 μm, 10 μm, or 8 μm. Includes dispersed phase droplets with an average diameter between 8 μm and 15 μm, 12 μm, or 10 μm; between 10 μm and 15 μm, or 12 μm; or between 12 μm and 15 μm.

In some embodiments, the food products provided herein do not use bulk suspension food products (ie, in colloid mills, ultrasonic crushers, rotor stator units). Emulsified food products (eg, salad dressings, mayonnaise), between 1 μm and 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, or 2 μm; 2 μm to 10 μm, 9 μm, 8 μm, 7 μm, 6 μm. Between 5 μm, 4 μm, or 3 μm; between 3 μm and 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, or 4 μm; between 4 μm and 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, or 5 μm; 5 μm to 10 μm, Between 9 μm, 8 μm, 7 μm, or 6 μm; between 6 μm and 10 μm, 9 μm, 8 μm, or 7 μm; between 7 μm and 10 μm, 9 μm, or 8 μm; between 8 μm and 10 μm, or between 9 μm; or between 9 μm and 10 μm. It is a dispersed phase droplet having an average diameter of.

Food Product Preparations There are various recipes for preparing food products, and any such recipe is used in the methods provided herein for producing the food products provided herein. You may. Fermented broths or preparations containing recombinant ingredients may be used in such recipes in place of or in addition to the commonly used food ingredients.

Although the present invention has been described in conjunction with a particular specific embodiment thereof, it should be understood that the above description is intended to be exemplary and does not limit the scope of the invention. Other aspects, advantages, and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention belongs.

The following examples are included to illustrate specific embodiments of the invention. The techniques disclosed in the examples represent techniques discovered by the inventors to function adequately in the practice of the invention; It should be understood that many modifications can be made in various embodiments and that similar or similar results are still obtained without departing from the spirit and scope of the invention. As a result, all matters described or shown in the examples should be judged as examples and are not in a limiting sense.

Removal of Esterase Activity Through Purification Recombinant Trichoderma reesei host cells capable of producing recombinant β-lactoglobulin are targeted and inducible for the protoplasts of the Trichoderma reesei strain for genomic integration. It was produced by transformation with a polynucleotide encoding β-lactoglobulin under the control of a promoter. Transformants were selected on media containing markers selected for incorporating recombinant polynucleotides.

Recombinant host cells were fermented in a culture medium suitable for the growth of recombinant host cells and the production and secretion of recombinant β-lactoglobulin.

Fermented broth was collected and the biomass and cell debris were removed by centrifugation to obtain purified fermented broth. Recombinant β-lactoglobulin was isolated from fermentation broth purified based on charge (ie, ionic complex formation), thermal instability / thermal stability, and / or hydrophobicity.

For example, recombinant β-lactoglobulin separates based on charge (ie, particles and ions having the opposite charge to recombinant β-lactoglobulin at a particular pH and / or ionic strength (eg, sodium acid salt). Complex formation followed by isolation of the complex and elution of recombinant β-lactoglobulin from the complex isolated via pH and / or ionic strength adjustment), followed by separation on a hydrophilic basis. (Ie, the phenylsefalose column is loaded with the recombinant β-lactoglobulin preparation at a suitable pH and / or ionic strength (eg, 0.8 M Na2SO4) to bind the esterase to phenylsefalose and dissolve in the flow-through. Isolated by allowing the collection of sex β-lactoglobulin). The β-lactoglobulin preparation was spray-dried as needed to give a β-lactoglobulin powder preparation.

In another example, recombinant β-lactoglobulin separates based on charge (ie, has the opposite charge to recombinant β-lactoglobulin at a particular pH and / or ionic strength (eg, acid sodium salt). Ionic complex formation with the particles, followed by isolation of the complex and elution of recombinant β-lactoglobulin from the complex isolated via pH and / or ionic strength adjustment), followed by thermal instability / heat. Separation based on stability (ie, with suitable pH and / or ionic strength (eg, pH 3 and conductivity below 2 mS / cm, eg, obtained by dialysis filtration)) and heating (eg, 80-85). (At ° C for 20-60 minutes), the esterase activity was precipitated, and then the precipitate was removed by centrifugation to obtain soluble β-lactoglobulin). The β-lactoglobulin preparation was spray-dried as needed to give a β-lactoglobulin powder preparation.

The esterase activity contained in the β-lactoglobulin preparation is such that the β-lactoglobulin powder preparation (3% w / w) is added to coconut and / or sunflower oil and homogenized at 200 bar to form an emulsion. It was decided by that. The mixture was assayed for the presence of free fatty acids using the FFA kit (Product No. KA1667; Abnova (Taipei City, Taiwan)).

As shown in FIGS. 1-4, the β-lactoglobulin preparation obtained by purification contained substantially removed esterase activity.

Removal of Esterase Activity Through Culture Recombinant Trichoderma reesei host cells capable of producing recombinant β-lactoglobulin are targeted for genomic integration of the Trichoderma reesei strain of protoplasts under the control of an inducible promoter. Generated by transformation with a polynucleotide encoding β-lactoglobulin in. Transformants were selected on media containing markers selected for incorporating recombinant polynucleotides.

Recombinant host cells were fermented in three cultures containing a culture medium suitable for the growth of recombinant host cells and the production and secretion of recombinant β-lactoglobulin. The three cultures differed from each other only in the type of antifoaming agent contained in the culture medium, which were selected from the list consisting of Industrol DF204, Polyglycol P-2000, and Erol DF6000K.

Fermented broth was collected from each culture and biomass and cell debris were removed by centrifugation to obtain purified fermented broth. The esterase activity contained in the purified fermentation broth is subjected to pNPL (Product No. 61716; Sigma-Aldrich, St. Louis, MO) and 4MUo assay (Product No. 75164; Sigma-Aldrich, St. Louis, MO). By doing so, it was basically decided for each manufacturer's protocol. For the 4Muo assay, 20 μL of purified fermented broth was combined with 0.1 M sodium acetate buffer pH 4.3, 20 μL, and 0.1 mM 4MU-o 40 μL in a white 96-well plate and the mixture was 60 at 43 ° C. After incubating for minutes, it was quenched at 0.1 M Borax solution pH 9.22, 80 μL and fluorescence was read by excitation at 365 nm and luminescence reading at 450 nm. For the pNPL assay, 40 μL of purified fermented broth at pH 4.3 is combined with 40 μL of 1 mM pNPL solution in a 96-well PCR plate, the mixture is incubated at 43 ° C. for 60 minutes and then it is equal volume 0.1 M. The Borax solution was quenched at pH 9.22 and the fluorescence was read by luminescence reading at 400 nm.

As shown in FIG. 5, purified fermented broth obtained from the culture utilized as the antifoaming agent Industrol DF204 or Polyglycol P-2000 by Chukey Kramer analysis was used as the antifoaming agent Erol DF6000K. It was demonstrated that it produced significantly lower esterase activity than the purified fermented broth obtained from the material.

Removal of Esterase Activity via Gene Modification Recombinant trichoderma lysee host cells capable of producing recombinant β-lactoglobulin and containing substantially removed cutinase (eg, cut1 (UniProt number G0RH85)) activity (eg, cut1 (UniProt number G0RH85)). Using a polynucleotide (target vector) genetically engineered to integrate "cutinase knockout recombinant host cells") by homologous recombination at the cutinase locus and to replace the cutinase open reading frame with a selectable marker. It was produced by transforming a protoplast of a trichoderma lyseee strain (“corresponding recombinant host cell”) capable of producing recombinant β-lactoglobulin.

The general structure of the target vector is shown in FIG. The target vector is a selective marker (pyr4 gene capable of growing without uracil addition) flanked by a polynucleotide sequence homologous to the upstream and downstream polynucleotide sequences flanking the cutinase open reading frame in the trichoderma lyseei genome. It was included. The upstream and downstream sequences are provided herein as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

Transformants were selected on minimal medium and then screened by PCR to identify cutinase knockout recombinant host cells. Cutinase knockout recombinant host cells and corresponding recombinant host cells were fermented in culture media suitable for the growth of recombinant host cells and the production and secretion of recombinant β-lactoglobulin. Fermented broth was collected and the biomass and cell debris were removed by centrifugation to obtain purified fermented broth. Recombinant β-lactoglobulin was isolated from fermented broth purified based on charge (ie, electrostatic interaction).

Cutinase activity in β-lactoglobulin preparations obtained from cutinase knockout strains and parent strains was assayed using the FFA assay.

As shown in FIG. 7, in the FFA assay, β-lactoglobulin preparations obtained from the corresponding recombinant host cells produce free fatty acids over time, which is the lipase in the β-lactoglobulin preparation. It is an indicator of the presence of activity. Such production of free fatty acids (ie, esterase activity) was substantially eliminated in the β-lactoglobulin preparation obtained from cutinase knockout recombinant host cells.

An ice cream composition comprising recombinant β-lactoglobulin produced in filamentous fungal cells and further comprising reduced esterase activity.
The following samples were prepared and incubated at 4 ° C. for 16 hours:

As shown in FIG. 9A, the use of recombinant β-lactoglobulin produced by filamentous fungal cells resulted in the formation of unwanted gel compositions. In addition, the preparation produced an unpleasant aroma and taste.

As shown in FIG. 9B, heat treatment of recombinant β-lactoglobulin prevented gelation (and reduced unpleasant aroma and taste production).

As shown in FIG. 9C, the addition of triacylglycerol lipase counteracts the effect of thermal treatment of recombinant β-lactoglobulin in that the preparation re-forms the gel composition (with an unpleasant aroma and taste). rice field.

As shown in FIG. 9D, the use of bovine β-lactoglobulin purified from whey did not provide an unwanted gel composition. It also did not produce an unpleasant aroma and taste.

As shown in FIG. 9E, addition of triacylglycerol lipase to bovine β-lactoglobulin resulted in gel formation (and produced an unpleasant aroma and taste).

As shown in FIG. 9F, the use of recombinant β-lactoglobulin preparations containing substantially eliminated esterase activity provided the desired ice cream texture with the desired aroma and taste.

Taken together, the data indicate that preparations of recombinant β-lactoglobulin produced by filamentous fungal cells may contain esterase activity that adversely affects the texture, aroma, and taste of the food composition. Suggest.

Identification of esterase activity that provides the desired functional profile Additional recombinant host cells capable of producing recombinant components (eg, recombinant milk proteins) (eg, additional recombinant Trichoderma lysee host cells, additional recombinants) The Aspergillus niger host cell, an additional recombinant Trichoderma citrinoviride host cell, and an additional recombinant Myceriophthora thermophila host cell are similar to those disclosed in Example 3. Esterase using a target vector containing upstream and downstream target sequences specific for the esterified esterase loci, or mutations (eg, alterations, deletions, or insertions of one or more nucleotides). An open reading encoding an esterase for introduction into a nucleotide sequence that regulates the expression of the esterase, or for producing recombinant host cells containing esterase activity that is modulated (ie, high or low) or substantially eliminated. Other esterases (eg, carboxyl ester hydrases (eg, UniProt number G0RBM4, G0RIU1), phosphorlipase C (eg, UniProt number G0REM9), acetylesterases (eg, UniProt number G0REM9), using other mutagenesis schemes for introduction into the frame. For example, UniProt number G0RHJ4), and transcription factors that regulate the expression of esterases (eg, UniProt number G0RX49), and any other esterase disclosed herein, two or more such other esterases. And one or more such other esterase-cutinase combinations (eg, UniProt No. G0RH85) are produced as described in Example 3. Is obtained from such recombinant host cells according to the methods described herein and is used to prepare food products. Food products have suitable esterase activity or suitable esterase activity that provides the food product with the desired functional profile. It is tested for the desired sensory properties for identifying the combination of esterase activities (eg, desired odor, desired taste, desired texture, desired emulsification). Suitable for providing the desired sensory profile for a particular food product. Esterase activity or d The combination of stellase activity may depend on the particular composition of the food product (eg, the particular lipid profile of the food product).

All publications, patents, patent applications, sequences, database entries, and other references mentioned herein are specific to individual references, patents, patent applications, sequences, database entries, or other references. It shall be incorporated by reference to the same extent as indicated specifically and individually. In the event of a conflict, the specification containing the definition will prevail. The terminology and description used herein are for purposes of illustration only and are not intended to limit the scope of the invention.

Claims (103)

Recombinant Microorganisms capable of producing recombinant components A method for producing food products containing recombinant components produced by host cells in which the activity of esterase is essentially eliminated or modulated. A method involving at least one step. The method according to claim 1, wherein the esterase is a single esterase. The method of claim 1, wherein the esterase comprises at least two esterases. The method of claim 1, wherein the esterase comprises a carboxyl ester hydrolase. The carboxyl ester hydrolase is the UniProt sequence G0RIU1, A2R1N7, G0RBM4, A0A2T4BBP9, G2Q379, A0A2T4AZ21, A2R8Z3, G0R9X3, A2QPC2, G0RCG3, A2QPC2, G0RCG3, A0A2T4BCL7. , The method according to claim 4. The method of claim 1, wherein the esterase comprises triacylglycerol lipase. The method of claim 6, wherein the triacylglycerol lipase comprises an amino acid sequence that is at least 80% identical to the sequence selected from the UniProt sequences A2QM14 and A2R709. The method of claim 1, wherein the esterase comprises phospholipase A2. The method of claim 8, wherein the phospholipase A2 comprises an amino acid sequence that is at least 80% identical to the UniProt sequence A2QIF8. The method of claim 1, wherein the esterase comprises lysophospholipase. The method of claim 10, wherein the lysophospholipase comprises an amino acid sequence that is at least 80% identical to a sequence selected from the UniProt sequences G0RX90, G2Q0K1, A0A2T4B235, A2QC75, and A2QC42. The method of claim 1, wherein the esterase comprises acetylesterase. 12. The method of claim 12, wherein the acetylesterase comprises an amino acid sequence that is at least 80% identical to the sequence selected from the UniProt sequences G0RHJ4, A0A2T4BJD9, and G2QL32. The method of claim 1, wherein the esterase comprises phospholipase C. 15. The method of claim 14, wherein the phospholipase C comprises an amino acid sequence that is at least 80% identical to the sequence selected from the UniProt sequences G0REM9, A0A2T4BFY3, and A2QAD7. The method of claim 1, wherein the esterase comprises cutinase. 16. The method of claim 16, wherein the cutinase comprises an amino acid sequence that is at least 80% identical to the sequence selected from the UniProt sequences G0RH85, A2QBP1, A0A2T4BJB5, A5ABE6, A2R2W3, and G2QH51. The method of claim 1, wherein the activity of the esterase is essentially eliminated. The method of claim 1, wherein the activity of the esterase is reduced. The method of claim 1, wherein the activity of the esterase is increasing. The method of claim 1, wherein said activity of the esterase comprises the activity of a first esterase that is essentially eliminated and the activity of a second esterase that is reduced. The method of claim 1, wherein the activity of the esterase comprises an essentially removed first esterase activity and an increased second esterase activity. The method of claim 1, wherein the activity of the esterase comprises a decreasing activity of the first esterase and an increasing activity of the second esterase. The at least one step in which the esterase activity is essentially eliminated or modulated cultivates the recombinant microbial host cell under conditions appropriate to essentially eliminate or modulate the esterase activity. The method of claim 1, comprising the step of performing. 25. The method of claim 25, wherein the appropriate conditions include the presence of an appropriate defoaming agent. The defoaming agents are ACP 1500, Antifoam 204, Erol DF6000K, Hodag K-60K, Industrol DF204, P-2000E, SAG 471, SAG 5693, SAG 710, SAG 730, Silicone defoaming agent, StruktolJ64 26. The method of claim 26, selected from the group consisting of and sunflower oil. The at least one step in which the esterase activity is essentially eliminated or modulated essentially eliminates or modulates said activity of the esterase as compared to the activity of esterase contained in the corresponding recombinant microbial host cell. The method of claim 1, comprising the step of producing the recombinant component in a recombinant microbial host cell comprising a gene modification to rate. The method according to claim 1, wherein the recombinant microbial host cell is a recombinant filamentous fungus host cell. The recombinant filamentous fungus host cell is derived from a group consisting of a recombinant black stag beetle host cell, a recombinant Myserioftora thermophila host cell, a recombinant trichoderma lyseei host cell, and a recombinant trichoderma citrinobilide host cell. , The method of claim 29. The method of claim 1, wherein the at least one step in which the esterase activity is essentially removed or modulated comprises the step of separating from the esterase and purifying the recombinant component. The method according to claim 1, wherein the recombinant component is a recombinant protein. 32. The method of claim 32, wherein the recombinant protein is selected from the group consisting of recombinant plant proteins, recombinant algae proteins, recombinant fungal proteins, recombinant microbial proteins, and recombinant animal proteins. 33. The method of claim 33, wherein the recombinant animal protein is a recombinant milk protein. 34. The method of claim 34, wherein the recombinant milk protein is a recombinant whey protein. 35. The method of claim 35, wherein the recombinant whey protein is selected from the group consisting of α-lactalbumin, β-lactoglobulin, lactoferrin, transferrin, serum albumin, lactoperoxidase, and glycomacropeptide. 34. The method of claim 34, wherein the recombinant milk protein is recombinant casein. 37. The method of claim 37, wherein the recombinant casein is selected from the group consisting of β-casein, γ-casein, κ-casein, α-S1 casein, and α-S2 casein. Claim 1 further comprises the step of purifying the recombinant component to obtain a preparation comprising the essentially removed or modulated activity of the esterase as compared to the recombinant component and the corresponding preparation. The method described in. The preparation imparts to the food product one or more properties selected from the group consisting of the desired flavor, desired aroma, desired texture, desired emulsion, desired nutritional content, and desired shelf life. 39. The method of claim 39, comprising an esterase activity profile optimized in such a manner. 39. The method of claim 39, wherein the preparation is a powder. Recombinant microorganisms capable of producing recombinant components Recombinant microorganisms comprising a genetic modification that essentially eliminates or modulates the activity or expression of esterase as compared to the activity or expression of esterase contained in the corresponding recombinant microorganism host cell. Host cell. The recombinant microbial host cell according to claim 42, wherein the esterase is a single esterase. The recombinant microbial host cell of claim 42, wherein the esterase comprises at least two esterases. The recombinant microbial host cell of claim 42, wherein the esterase comprises a carboxyl ester hydrolase. The carboxyl ester hydrolase is the UniProt sequence G0RIU1, A2R1N7, G0RBM4, A0A2T4BBP9, G2Q379, A0A2T4AZ21, A2R8Z3, G0R9X3, A2QPC2, G0RCG3, A2QPC2, G0RCG3, A0A2T4BCL7. 45. The recombinant microbial host cell according to claim 45. The recombinant microbial host cell of claim 42, wherein the esterase comprises triacylglycerol lipase. The recombinant microbial host cell of claim 47, wherein the triacylglycerol lipase comprises an amino acid sequence that is at least 80% identical to the sequence selected from the UniProt sequences A2QM14 and A2R709. The recombinant microbial host cell of claim 42, wherein the esterase comprises phospholipase A2. The recombinant microbial host cell of claim 49, wherein the phospholipase A2 comprises an amino acid sequence that is at least 80% identical to the UniProt sequence A2QIF8. The recombinant microbial host cell of claim 42, wherein the esterase comprises lysophospholipase. The recombinant microbial host cell of claim 51, wherein the lysophospholipase comprises an amino acid sequence that is at least 80% identical to a sequence selected from the UniProt sequences G0RX90, G2Q0K1, A0A2T4B235, A2QC75, and A2QC42. The recombinant microbial host cell of claim 42, wherein the esterase comprises acetylesterase. The recombinant microbial host cell of claim 53, wherein the acetylesterase comprises an amino acid sequence that is at least 80% identical to the sequence selected from the UniProt sequences G0RHJ4, A0A2T4BJD9, and G2QL32. The recombinant microbial host cell of claim 42, wherein the esterase comprises phospholipase C. The recombinant microbial host cell of claim 55, wherein the phospholipase C comprises an amino acid sequence that is at least 80% identical to the sequence selected from the UniProt sequences G0REM9, A0A2T4BFY3, and A2QAD7. The recombinant microbial host cell of claim 42, wherein the esterase comprises cutinase. 58. The recombinant microbial host cell of claim 57, wherein the cutinase comprises an amino acid sequence that is at least 80% identical to the sequence selected from the UniProt sequences G0RH85, A2QBP1, A0A2T4BJB5, A5ABE6, A2R2W3, and G2QH51. The recombinant microbial host cell of claim 42, wherein the activity of the esterase is essentially eliminated. The recombinant microbial host cell of claim 42, wherein the activity of the esterase is reduced. The recombinant microbial host cell of claim 42, wherein the activity of the esterase is increasing. 42. The recombinant microbial host cell of claim 42, wherein said activity of the esterase comprises the activity of a first esterase that is essentially eliminated and the activity of a second esterase that is reduced. 42. The recombinant microbial host cell of claim 42, wherein said activity of the esterase comprises the activity of a first esterase that is essentially eliminated and the activity of a second esterase that is increased. 42. The recombinant microbial host cell of claim 42, wherein the activity of the esterase comprises a decreasing activity of the first esterase and an increasing activity of the second esterase. The genetic modification is a gene modification in a regulatory sequence or a functional portion thereof that promotes the expression of the esterase or a protein required for the expression of the esterase, and a code encoding a protein required for the expression of the esterase or a functional portion thereof. Gene modification in the sequence, gene modification in the coding sequence encoding the esterase or its functional part, gene modification in the regulatory sequence or its functional part that promotes the expression of the endogenous inhibitor of the esterase, endogenous inhibition of the esterase. The recombinant microbial host cell according to claim 42, which is selected from the group consisting of a gene modification in a coding sequence encoding a factor and a gene modification introducing a coding sequence encoding a heterologous inhibitor of the esterase. The genetic modification is a genetic modification in a regulatory sequence or functional portion thereof that promotes expression of a transcription factor that regulates the expression of the esterase, or in a coding sequence that encodes such a transcription factor or functional portion thereof. 42. The recombinant microbial host cell according to claim 42. The recombinant host according to claim 66, wherein the transcription factor comprises an amino acid sequence that is at least 80% identical to a sequence selected from the UniProt sequences G0RX49, G0RHG1, A0A2T4B416, A0A2T4BJU6, A2R2J1, A2R903, G2Q2Z5, and G2Q8I6. cell. The recombinant microbial host cell according to claim 42, which is a recombinant filamentous fungus host cell. The recombinant filamentous fungus host cell is selected from the group consisting of a recombinant black stag beetle host cell, a recombinant Myserioftora thermophila host cell, a recombinant trichoderma lyseei host cell, and a recombinant trichoderma citrinobilide host cell. The recombinant microbial host cell according to claim 68. The recombinant microbial host cell according to claim 42, wherein the recombinant component is a recombinant protein. The recombinant microbial host cell according to claim 70, wherein the recombinant protein is selected from the group consisting of a recombinant plant protein, a recombinant algae protein, a recombinant fungal protein, a recombinant microbial protein, and a recombinant animal protein. .. The recombinant microbial host cell according to claim 71, wherein the recombinant animal protein is a recombinant milk protein. The recombinant microbial host cell according to claim 72, wherein the recombinant milk protein is a recombinant whey protein. 23. The recombinant microbial host according to claim 73, wherein the recombinant whey protein is selected from the group consisting of α-lactalbumin, β-lactoglobulin, lactoferrin, transferrin, serum albumin, lactoperoxidase, and glycomacropeptide. cell. The recombinant microbial host cell according to claim 72, wherein the recombinant milk protein is recombinant casein. The recombinant microbial host cell according to claim 75, wherein the recombinant casein is selected from the group consisting of β-casein, γ-casein, κ-casein, α-S1 casein, and α-S2 casein. Recombinant Microbial A food product containing a recombinant component produced by a host cell that further comprises the activity of an esterase that is essentially eliminated or modulated relative to the activity of the esterase in the corresponding food product. .. The food product according to claim 77, wherein the esterase is a single esterase. The food product according to claim 77, wherein the esterase comprises at least two esterases. The food product according to claim 77, wherein the activity of the esterase is essentially nonexistent. The food product according to claim 77, wherein the activity of the esterase is reduced. The food product of claim 77, wherein the activity of the esterase is increasing. The food product according to claim 77, which is a dairy product substitute. The food product according to claim 83, wherein the dairy substitute is a dairy substitute selected from the group consisting of milk, yogurt, cheese, daily spreads, creams, frozen desserts, butter, and dairy powder. The food product according to claim 77, which comprises a milk protein component. The food product of claim 85, wherein the milk protein component comprises at least one recombinant milk protein. The food product of claim 85, wherein the at least one recombinant milk protein is a single recombinant milk protein. The food product according to claim 87, wherein the single recombinant milk protein is recombinant β-lactoglobulin. The food product of claim 87, wherein the single recombinant milk protein is recombinant α-lactalbumin. The food product of claim 87, wherein the single recombinant milk protein is recombinant κ-casein. The food product of claim 87, wherein the single recombinant milk protein is recombinant β-casein. The food product of claim 87, wherein the single recombinant milk protein is recombinant γ-casein. The food product of claim 85, wherein the at least one recombinant milk protein is two recombinant milk proteins. The food product according to claim 93, wherein the two recombinant milk proteins are recombinant β-lactoglobulin and recombinant α-lactalbumin. The food product of claim 93, wherein the two recombinant milk proteins are recombinant κ-casein and recombinant β-casein. The food product of claim 93, wherein the two recombinant milk proteins are recombinant κ-casein and recombinant γ-casein. The food product according to claim 93, wherein the two recombinant milk proteins are recombinant β-casein and recombinant γ-casein. The food product of claim 93, wherein the two recombinant milk proteins are recombinant β-lactoglobulin and recombinant κ-casein. The food product of claim 93, wherein the two recombinant milk proteins are recombinant κ-casein and recombinant α-lactalbumin. The food product according to claim 93, wherein the two recombinant milk proteins are recombinant β-lactoglobulin and recombinant β-casein. The food product of claim 93, wherein the two recombinant milk proteins are recombinant β-casein and recombinant α-lactalbumin. The food product of claim 93, wherein the two recombinant milk proteins are recombinant β-lactoglobulin and recombinant γ-casein. The food product of claim 93, wherein the two recombinant milk proteins are recombinant γ-casein and recombinant α-lactalbumin. 17. The food product of claim 77, comprising a free lipid content that is within the desired range to give the food product the desired sensory profile.

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